Artificial blood vessel and method of manufacturing thereof

ABSTRACT

An artificial tissue capable of carrying necessary nutrients for maintaining activities of cells and tissues, and a method of manufacturing an artificial blood vessel. A plurality of forms of blood vessels are extracted from an image of a living tissue and made into a blood vessel form image. Each of the blood vessel forms of the blood vessel form image is adjusted and a blood vessel formation pattern is formed. A blood vessel cell culturing pattern of forming is formed, in a cell culturing layer. The blood vessel cell culturing pattern includes: a cell adhesion portion having adhesive properties with a blood vessel cell and formed to the blood vessel formation pattern; and a cell adhesion-inhibiting portion having cell adhesion-inhibiting properties for inhibiting adhesion with a blood vessel cell and formed in an area other than the cell adhesion portion. A blood vessel cell is adhered to the cell adhesion portion, and cultured into a tissue.

TECHNICAL FIELD

The present invention relates to an artificial blood vessel used in thefield of the regenerative medicine or the like.

BACKGROUND ART

At present, cell cultures of various animals and plants are performed,and also new cell culture methods are in development. The technologiesof the cell culture are utilized, such as to elucidate the biochemicalphenomena and natures of cells and to produce useful substances.Furthermore, with cultured cells, an attempt to investigate thephysiological activity and toxicity of artificially synthesized medicalis under way. Moreover, in the field of the medicine and others,artificial production of tissues and organs has been attempted byre-organizing such as cells, proteins, glucides, or lipids of livingbodies by the technique of the cell engineering, or the like.

Here, since the common animal cells perish without supply of thenutrients, or the like, in the case of using cultured cells as, forexample, the artificial tissues, it is necessary to provide thecapillary blood vessels in the artificial tissues and the blood forpassing through therein for supplying such as the oxygen or nutrients,and carrying out the metabolic decomposition products. Moreover,disorders, in which an infarct in micro blood vessels within the livingbody occurs, and the supply of oxygen and nutrients or the transfer ofmetabolic decomposition products are not sufficiently performed exist.

To respond to such problems, conventionally, for example, artificialformation of the capillary blood vessels has been attempted by thetechniques of the Non-Patent Documents 1 to 3, however, in either case,only the vessel-like tissues (capillaries) are formed in disorder sothat it has been difficult to form capillary blood vessels capable ofproviding a necessary amount of the blood to a desired position formaintaining the function of the artificial tissues or infarct sitetreatment of micro blood vessels. Moreover, as described in Non-PatentDocument 4 or 5, a forming method of blood vessels by utilizing anartificial material has been studied, however, it is difficult to formnarrow blood vessels and the artificial blood vessels which can beutilized for infarct site treatment of micro blood vessels and theconstruction of an artificial tissue could not be formed.

On the other hand, the present inventors have proposed a method ofculturing cells in a pattern by changing the surface of a layer havingcell adhesive properties or cell adhesion-inhibiting properties by thefunction of a photocatalyst accompanied by the irradiation with energyfor forming a pattern comprising a cell adhesion portion and a celladhesion-inhibiting portion and highly accurately adhering the cellsonly to the cell adhesion portion. According to the patterning method,the cells are stimulated at the boundary of the cell adhesion portionand the cell adhesion-inhibiting portion so that the cells adhered in apattern can be aligned or the morphological change to the stretchingstate can be promoted strongly as a result. Using the present method, itbecame possible that by culturing vascular endothelial cells in apattern form and further by making an anchorage material for derivingthe vascular endothelial cells into a blood vessel tissue in contactwith the endothelial cells and transcribing the cells, a narrow bloodvessel tissue along the desired pattern is formed. However, thetechnology for forming these blood vessels in a form suitable for theinfarction site treatment of the micro blood vessels and theconstruction of the above-described artificial tissue has not yet beeninvented.

-   [Non-Patent Documents 1] D. E. Ingber, et al., The Journal of Cell    Biology (1989) p. 317—-   [Non-Patent Documents 2] B. J. Spargo, et al., Proceedings of the    National Academy of Sciences of the United States of    America (1994) p. 11070—-   [Non-Patent Documents 3] R. Auerbach et al., Clinical    Chemistry (2003) p. 32—-   [Non-Patent Documents 4] C. B. Weinberg, et al., Science (1986) p.    397—-   [Non-Patent Documents 5] N. L'. Heureux, et al., The FASEB    Journal (1998) vol. 12 p. 47—

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

From the descriptions set forth above, it is necessarily required thatoxygen and nutrients necessary for maintaining the functions areefficiently supplied and a metabolic decomposition product is sent outfor the purpose of treating the infarct site of micro blood vessels andconstructing an artificial tissue and organ. Accordingly, it has beendesired that a method of manufacturing an artificial blood vessel formedin a form suitable for the infarct site treatment of micro blood vesselsand an artificial tissue constructed outside of the living body isprovided.

Means for Solving the Problem

The present invention provides a method of manufacturing an artificialblood vessel, characterized in that the method comprises: a process forextracting a blood vessel form image of extracting a plurality of formsof blood vessels from an image of a living tissue and making it into ablood vessel form image; a process for adjusting a blood vessel formingpattern of adjusting each of the blood vessel forms of the blood vesselform image and forming a blood vessel formation pattern; a process forforming a blood vessel cell culturing pattern of forming, in a cellculturing layer, a blood vessel cell culturing pattern comprising: acell adhesion portion having adhesive properties with a blood vesselcell and formed to the blood vessel formation pattern, and a celladhesion-inhibiting portion having cell adhesion-inhibiting propertiesfor inhibiting adhesion with a blood vessel cell and formed in an areaother than the cell adhesion portion; and a process for culturing ablood vessel cell of adhering a blood vessel cell to the cell adhesionportion, culturing and made into a tissue.

As a form of artificial blood vessels suitable for the infarction sitetreatment of micro blood vessels and an artificial tissue constructedoutside of the living body, there is a form mocking the structure ofblood vessels in a tissue of the living body. In a tissue of the livingbody, oxygen, nutrients and metabolic decomposition products which arenecessary for cells in the tissue to activate are carried via blood.

According to the present invention, since in a process for forming theabove-described blood vessel cell culturing pattern, the above describedcell adhesion portion which has been adjusted so that the blood vesselcell can form blood vessels in a blood vessel pattern form of the livingbody tissue is formed, it becomes possible to form artificial bloodvessels in a pattern form similar to the blood vessels of the livingbody tissue in a process for culturing blood vessel cell. Owing to this,it is capable of, for example, supplying nutrients from the bloodvessels to cells in a tissue similar to the case of the tissue of theliving body by disposing artificial blood vessels manufactured accordingto the present invention at the infarction site of micro blood vesselsor in the artificial tissue. Moreover, in the present invention, a bloodvessel form image obtained from the image of the living body tissue isadjusted in the above-described process for adjusting a blood vesselforming pattern, it becomes possible that the blood vessels areefficiently formed in a pattern form to be targeted by utilizing theabove-described process for forming a blood vessel cell culturingpattern and the process for culturing a blood vessel cell.

In the above-described invention, the above-described process forforming a blood vessel cell culturing pattern can be made into a processfor forming blood vessel cell culturing pattern by irradiating theenergy to the above-described cell culturing layer which can form theabove-described cell adhesion portion and the above-described celladhesion-inhibiting portion by action of a photocatalyst accompanyingwith the energy irradiation. In this case, it has an advantage that atthe time when the above-described blood vessel cell culturing pattern isformed, blood vessel cell culturing pattern can be formed easily and ata finely processed level without utilizing complex processes such as aprocess for utilizing chemicals which have an adverse influence upon thecell.

According to the present invention, a method of manufacturing anartificial tissue characterized in that it utilizes the artificial bloodvessel manufactured by the above-described blood vessel manufacturingmethod is provided. According to the present invention, since artificialblood vessels manufactured by the above-described manufacturing methodis used, nutrients can be supplied to cells in the manufacturedartificial tissue similar to the tissue of the living body and so on,and the artificial tissue can be used for a variety of uses.

The present invention further provides a photomask, characterized inthat the photomask has a blood vessel pattern comprising atwo-dimensional pattern constituted with a line width in which avascular endothelial cell is in a tubular form.

According to the present invention, since a photomask has a blood vesselpattern comprising a two-dimensional pattern constituted by the linewidth in which the vascular endothelial cell is in a tubular form, byutilizing this photomask, for example, a cell adhesion portion havingthe adhesive properties with a cell is formed in a blood vessel form ina living body tissue by patterning the cell culturing layer, and thevascular endothelial cells can be cultured on the cell adhesion portionand made these into a tubular form. Owing to this, the blood vessels canbe formed into a targeted pattern, and for example, artificial bloodvessels having a pattern similar to the blood vessels of the living bodytissue can be formed.

The present invention further provides an artificial blood vessel,characterized in that the artificial blood vessel has a blood vesselpattern formed by a two-dimensional pattern constituted with a linewidth in which a vascular endothelial cell is in a tubular form.

Since artificial blood vessels of the present invention have a bloodvessel pattern formed by utilizing a two-dimensional pattern constitutedby a line width in which a vascular endothelial cell is to be a tubularform, for example, it can be made artificial blood vessels which can beapplied for a variety of uses such as for a living tissue grafting, orfor artificial tissue.

The present invention further provides an artificial tissue,characterized by comprising the artificial blood vessel.

Since an artificial tissue of the present invention has artificial bloodvessels having a form of blood vessels of a living tissue, it can supplynutrients to the cells similar to the living tissue cell so that it canbe used for a variety of uses.

Moreover, the present invention provides a blood vessel cell culturingpattern base material comprising: a base material; a cell culturinglayer formed on the base material and having a pattern comprising a celladhesion portion having adhesive properties with a blood vessel cell anda cell adhesion-inhibiting portion for inhibiting adhesion with a bloodvessel cell; and a blood vessel cell adhered to the cell adhesionportion, characterized in that the cell adhesion portion is formed in ablood vessel pattern comprising a two-dimensional pattern constitutedwith a line width in which a vascular endothelial cell is in a tubularform.

According to a blood vessel cell culturing pattern base material of thepresent invention, these can be cultured in a finely processed patternform that the blood vessel cells are targeted on the above-describedcell adhesion portion.

The present invention further provides a vascular endothelial cellpattern base material comprising a base material and a vascularendothelial cell provided on the base material in such a way that thecell can be peeled off, characterized in that the vascular endothelialcell is formed with a line width in which the endothelial cell is in atubular form in a pattern that a blood vessel network is represented intwo-dimension.

In the vascular endothelial cell pattern base material of the presentinvention, since a vascular endothelial cell formed in a predeterminedpattern has been provided in a state where the vascular endothelial cellcan be peeled off, it can be used for a variety of uses, for example,for artificial tissue by peeling off the vascular endothelial cell.

Effect of the Invention

According to the present invention, it is possible that, for example,nutrients can be supplied to a cell in a tissue similar to a livingtissue, the present invention exerts the effect that artificial bloodvessels capable of being used for a variety of uses can be efficientlyformed in a pattern in which artificial blood vessels are targeted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C are each a photograph and illustrations for explainingone example of a method of manufacturing an artificial blood vessel ofthe present invention;

FIGS. 2A and 2B are illustrations for explaining one example of a methodof manufacturing an artificial blood vessel of the present invention;

FIGS. 3A and 3B are illustrations for explaining one example of aprocess for forming a blood vessel cell culturing pattern in amanufacturing method of an artificial blood vessel of the presentinvention;

FIGS. 4A and 4B are illustrations for explaining one example of aprocess for forming a blood vessel cell culturing pattern in amanufacturing method of an artificial blood vessel of the presentinvention; and

FIG. 5 shows a schematically sectioned view for illustrating one exampleof a blood vessel cell culturing pattern base material of the presentinvention.

DESCRIPTION OF REFERENCE NUMERALS

-   1 . . . cell culturing layer,-   2 . . . cell adhesion portion, and-   3 . . . cell adhesion-inhibiting portion.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to an artificial blood vessel used such asin the field of regenerative medicine and a manufacturing methodthereof, an artificial tissue in which the artificial blood vessels havebeen used and the manufacturing method thereof, a photomask which isused at time when these are manufactured, and a blood vessel cellculturing pattern base material forming the artificial blood vessel.Hereinafter, these will be explained in detail, respectively.

A. A Method of Manufacturing an Artificial Blood Vessel

First, the method of manufacturing an artificial blood vessel will beexplained. The method of manufacturing an artificial blood vessel of thepresent invention is characterized in that the method comprises: aprocess for extracting a blood vessel form image of extracting aplurality of forms of blood vessels from an image of a living tissue andmaking it into a blood vessel form image; a process for adjusting ablood vessel forming pattern of adjusting each of the blood vessel formsof the blood vessel form image and forming a blood vessel formationpattern; a process for forming a blood vessel cell culturing pattern offorming, in a cell culturing layer, a blood vessel cell culturingpattern comprising: a cell adhesion portion having adhesive propertieswith a blood vessel cell and formed to the blood vessel formationpattern, and a cell adhesion-inhibiting portion having celladhesion-inhibiting properties for inhibiting adhesion with a bloodvessel cell and formed in an area other than the cell adhesion portion;and a process for culturing a blood vessel cell of adhering a bloodvessel cell to the cell adhesion portion, culturing and made into atissue.

A method of manufacturing an artificial blood vessel of the presentinvention has, for example, as shown in FIGS. 1A to 1C, a process forextracting a blood vessel form image in which a form of blood vessels isextracted from an image of a living tissue (FIG. 1A) and made it intothe blood vessel form image (FIG. 1B); a process for adjusting a bloodvessel forming pattern in which the respective blood vessel forms of theblood vessel form image (FIG. 1B) and made it into the blood vesselforming pattern (FIG. 1C); for example as shown in FIGS. 2A and 2B, aprocess for forming a blood vessel cell culturing pattern (FIG. 2A) forforming a blood vessel cell culturing pattern on the cell culturinglayer 1 comprising a cell adhesion portion 2 which has been formed inthe above-described blood vessel forming pattern, and a celladhesion-inhibiting portion 3 which is the area other than the celladhesion portion 2; and a process for culturing a blood vessel cell(FIG. 2B) in which a blood vessel cell 4 is adhered to the cell adhesionportion 2 of the blood vessel cell culturing pattern and cultured andmade into a tissue.

A living tissue as used herein refers to a tissue formed by bloodvessels and the other cells and the like existing in the living body andalso means various kinds of organs such as kidney and liver, ocularfundus, and skin.

According to the present invention, in the process for forming bloodvessel cell culturing pattern, since a cell adhesion portion having thecell adhesive properties with a blood vessel cell is formed, on the cellculturing layer, in a form of a blood vessel forming pattern which is apattern similar to blood vessels in the living tissue, in the processfor culturing a blood vessel cell, the blood vessel cell can be adheredonly to the cell adhesion portion and cultured, and not adhered to thecell adhesion-inhibiting portion. Owing to this, it becomes possiblethat artificial blood vessels are formed in the pattern similar to theblood vessels of the living tissue. Accordingly, nutrients can besupplied similarly to the living tissue, for example, by disposing theartificial blood vessel in the tissue cell such as at the infarct siteof micro blood vessels, or in an artificial tissue.

Moreover, in the present invention, since the blood vessel form imagederived from the image of the living tissue obtained in the process foradjusting a blood vessel forming pattern is adjusted into a form inwhich artificial blood vessels are easily formed and so on in the bloodvessel cell culturing pattern forming process and the blood vessel cellculturing process, it becomes possible that artificial blood vessels areefficiently formed into a targeted pattern, and it is also preferablefrom the viewpoint of such as manufacturing efficiency.

Hereinafter, the respective processes of a method of manufacturing anartificial blood vessel of the present invention will be explained indetail.

1. Blood Vessel Forming Image Extracting Process

First, the blood vessel forming image extracting process of the presentinvention will be explained below. The blood vessel forming imageextracting process of the present invention is a process of extracting aform of blood vessels from the image of the living tissue to make ablood vessel form image. The method is not particularly limited as longas it is a method capable of obtaining an image extracting the form ofblood vessels from the image of the living tissue.

As an image of the living tissue used in the present process, it is notparticularly limited as long as the image is an image that the tissuewithin the living body has been imaged and that can specify the form ofthe blood vessels. As the image, for example, an image which has beenthree-dimensionally shot may be the one, however it is particularlypreferable that it is an image which has been two-dimensionally shot.This is because the adjustment in the process for adjusting a bloodvessel forming pattern described later becomes easy by utilizing animage which has been two-dimensionally shot. As a method of shooting theimage, for example, a method of directly observing blood vessels in theliving tissue as shown in blood vessels of skin or ocular fundus, amethod of directly shooting the cross-section of the living tissue, amethod of imaging it by injecting a contrast and opaque media into theblood vessels of the targeted living tissue and irradiating nuclearradiation, or a method of using MRI (magnetic resonance imaging) can beused.

In the present process, from an image of the living tissue describedabove, only the image of blood vessels is extracted. Most of theabove-described method of acquiring an image of blood vessels containsnoise or the like derived from the tissue other than blood vessels.Accordingly, as a method of extracting only the image of the bloodvessels, in order to remove the contour of the living tissue and thenoise, it is general that a binary treatment and a treatment ofexpanding and retracting an image are performed with respect to theobtained image to take out an image of blood vessels.

2. Blood Vessel Formation Pattern Adjusting Process

Next, the blood vessel formation pattern adjusting process in thepresent invention will be explained below. The blood vessel formationpattern adjusting process in the present invention is a process in whichthe forms of the respective blood vessels of the blood vessel formimages extracted by the process for extracting a blood vessel form imageis adjusted to form a blood vessel forming pattern. In the presentprocess, the form of the artificial blood vessels formed by the processfor forming a blood vessel cell culturing pattern and the process forculturing a blood vessel cell described later is to be determined byadjusting the form of the blood vessels of a blood vessel form image.

As an adjustment of a form of the respective blood vessels in thepresent process, for example, the adjustment of the line width of theblood vessels in the above-described blood vessel form image can belisted. Since it can be made that artificial blood vessels can beefficiently formed by adjusting the width of the respective bloodvessels of the blood vessel form image to a width suitable for formingartificial blood vessels in the blood vessel cell culturing patternforming process and the blood vessel forming pattern adjusting process;that is the line width in which a vascular endothelial cell is in atubular form. As the line width of blood vessels of a blood vessel cellculturing pattern formed in the process for forming a blood vessel cellculturing pattern described later using the above-described blood vesselforming pattern, it is preferable to be usually in the range from 20 μmto 100 μm, more preferable to be in the range from 40 μm to 80 μm, andit is particularly preferable to be in the range from 50 μm to 70 μm. Inthe case where the diameter of the blood vessel in the blood vessel formimage is smaller than the above-described range, it is desired that theblood vessel cell culturing pattern is changed into the suitablediameter for forming the blood vessels. It should be noted that in thecase where the diameter of the blood vessels in the blood vessel formimage is larger than the above-described range and so on, for example,it might be made into a pattern of several pieces of blood vesselsbranched in the above-described range to secure the necessary flow ofthe blood. At this time, usually, the line width of the respective bloodvessels is formed so as to be the same line width. And further, in thecase where the diameter of the blood vessel in the blood vessel formimage is larger than the above-described range, an auxiliary pattern maybe formed in the blood vessel pattern. The auxiliary pattern is a finepattern formed within the blood vessel pattern, and the area where thisauxiliary pattern has been formed is made into a celladhesion-inhibiting portion for inhibiting the adhesion with a bloodvessel cell in a blood vessel cell culturing pattern formed by theprocess for forming a blood vessel cell culturing pattern describedlater. It should be noted that, when the blood vessel cells are adheredto the blood vessel cell adhesion portion formed in pattern, theauxiliary pattern is formed in such a degree that the celladhesion-inhibiting portion corresponding to the auxiliary patternbecomes a fine pattern which does not inhibit the binding of the bloodvessel cells within the cell adhesion portion; specifically, in such adegree that the blood vessel cells can be bound with one another even onthe cell adhesion-inhibiting portion corresponding to the auxiliarypattern.

Generally, in the case where a tissue is formed by adhering a bloodvessel cell to the cell adhesion portion corresponding to the bloodvessel pattern and culturing the cell, the blood vessel cells aregradually aligned from the outside to the inside of the cell adhesionportion. Moreover, at the time when the tissue is formed, it isnecessary to align the blood vessel cells after the forms of therespective blood vessel cell are changed, and also concerning with thechanging of the form of the blood vessel cells, it is graduallyperformed from the end portion to the central portion of the celladhesion portion. Therefore, in the case where the width of the celladhesion portion is large, the alignment property of the blood vesselcells is poor at the central portion of the cell adhesion portion, andthere are cases where the tissue is not formed, where the blood vesselcells are adhered to the central portion of the cell adhesion portionand the like. Accordingly, since it becomes possible that the bloodvessel cells are aligned from the end portion of the celladhesion-inhibiting portion and the form can be changed by forming thecell adhesion-inhibiting portion corresponding to the auxiliary pattern,the blood vessel cells can be cultured in a targeted pattern formwithout generating the deletion and the failure in the form change.Moreover, since the cell adhesion-inhibiting portion corresponding tothe auxiliary pattern is formed so that the adhesion between the bloodvessel cells adjoining to one another sandwiching the celladhesion-inhibiting portion is not inhibited, as the width of the bloodvessel cells which are to be finally cultured, it can be the widthsimilar to the width corresponding to the pattern of the above-describedblood vessels.

Here, it is preferable that the auxiliary pattern is formed in a lineform within the pattern of the blood vessels. Moreover, the form of theline is not particularly limited and it can be made, for example, into alinear form, curve form, dotted form, and broken line form. It ispreferable that the line width of the auxiliary pattern is formed suchthat the width of the corresponding cell adhesion-inhibiting portionbecomes in the range from 0.5 μm to 10 μm, particularly in the rangefrom 1 μm to 5 μm at the time when the blood vessel cell culturingpattern was formed. In the case where the width is wider than theabove-described range, it is not preferable since it becomes difficultfor blood vessel cells adjoining to one another sandwiching the celladhesion-inhibiting portion corresponding to the auxiliary pattern tointeract on the cell adhesion-inhibiting portion.

Moreover, the auxiliary pattern may be formed to have a convexvoncavopattern such as a zig-zag form within the surface. At this time, it maybe formed such that the average value of the distance from the end ofthe convex portion to the end of the concave portion of theconvexvoncavo pattern becomes a distance in which the blood vessel cellsare lined-up in a direction similar to the line direction of the celladhesion portion at the time when the blood vessel cells were adhered tothe cell adhesion portion corresponding to the pattern of theabove-described blood vessels. It is preferable that it is formed suchthat the convexvoncavo is in the range form 0.5 μm to 30 μm particularlyat the time when the blood vessel cell culturing pattern described laterwas formed. It should be noted that as for the measurement of theaverage distance from the end of the concave portion to the end of theconvex portion of the convexvoncavo, the distance from the bottomportion to the apex portion of the respective convexvoncavo in the rangeof the length of 200 μm of the end portion of the celladhesion-inhibiting portion corresponding to the auxiliary pattern ismeasured, and it is made a value that the average was calculated.

Moreover, in the present process, it is preferable that the adjustmentfor amending the unclear portion and minute portion is performed. Thisis because it becomes difficult to form the blood vessels in a targetedpattern form in the process for forming a blood vessel cell culturingpattern and the process for culturing blood vessel cells described laterin the case where the blood vessel cell culturing pattern has theunclear portion and the like. Moreover, at this time, it may be adjustedby removing the unnecessary blood vessels. This is because in the casewhere the form of the blood vessels not existing on the same plane is inthe blood vessel form image, for example, in the case where the bloodvessel form image is an image three-dimensionally shot and so on, it ispreferable to remove this portion.

Moreover, since the blood vessel form image which exists inthree-dimension is expressed in two-dimension, although the intervalbetween the blood vessels is originally wide, there are a case where theinterval is expressed as it is narrow, or a case where the angle of thebranch is expressed to be more acute angle m than the original angle. Inthe above-described cases, in the present process, the adjustment thatwidens the interval between the blood vessels and the adjustment to makethe angle of the branch obtuse angle, and the like can be performed.Furthermore, in order to express the three-dimensional blood vesselnetwork in two-dimension where the blood vessels do not cross, theadjustment for exchanging the end portion of one of the crossed bloodvessels can be performed. Moreover, further, in the case where the bloodflow of the blood vessels can be made better without damaging thefunction that the blood vessels carries out and so on, the form of theblood vessels may be adjusted, for example, by making the blood vesselsformed in a curve form in the living tissue linear form.

Moreover, at the time when the blood vessels manufactured by the presentinvention are used for the living body and artificial tissue and so on,since it is naturally a state where the blood is flown in the bloodvessels and these are used, the adjustment maybe formed so that itbecomes a pattern such that it has the blood vessels not existing in theblood vessel form image for the purpose of communicating the bloodvessels in the living body and the respective blood vessels. As for theblood vessels described above, the diameter and the location where it isformed are appropriately adjusted by such as the direction where theblood flows or the pressure applied to the blood vessels.

The adjustment of the respective blood vessels as described above can beperformed by image treating a blood vessel form image and so on.

3. Blood Vessel Cell Culturing Pattern Forming Process

Next, the blood vessel cell culturing pattern forming process in thepresent invention will be explained below. The blood vessel cellculturing pattern forming process in the present invention is a processof forming, in the cell culturing layer, a blood vessel cell culturingpattern comprising: the cell adhesion portion having adhesive propertieswith a blood vessel cell and formed to a form of the above-describedblood vessel forming pattern; and the cell adhesion-inhibiting portionhaving the cell adhesion-inhibiting properties for inhibiting theadhesion with the blood vessel cells and formed in the area other thanthe cell adhesion portion. It should be noted that at the time when theblood vessel cell culturing pattern is formed using a blood vesselforming pattern, the size of the above-described cell culture portionmay be made the size same with the blood vessel forming pattern.Alternatively, the cell adhesion portion may be formed such that thesize of the blood vessel forming pattern is expanded or retracted. Owingto this, because it becomes possible that the blood vessel cellculturing pattern having a variety of sizes is formed by means of theone blood vessel forming pattern.

In the present process, by forming the blood vessel cell culturingpattern, in the cell culturing layer, comprising the cell adhesionportion and the cell adhesion-inhibiting portion, it becomes possiblethat the blood vessel cells are easily cultured in the blood vesselforming pattern by the process for culturing blood vessel cellsdescribed later. Here, to have the adhesive properties with blood vesselcells means that it excellently adheres to the blood vessel cells; andin the case where the adhesive properties with the blood vessel cells isdifferent according to the kinds of the blood vessel cells and so on, itmeans that it excellently adheres to the targeted blood vessel cells.Moreover, to have the cell adhesion-inhibiting properties with the bloodvessel cells means that it has the nature for inhibiting the adhesion ofthe blood vessel cells; and in the case where the celladhesion-inhibiting properties with the blood vessel cells are differentaccording to the kinds of the blood vessel cells and so on, it meansthat the adhesion with the targeted blood vessel cells is inhibited.

As for cell culturing layer used in the present process, if it is alayer which is capable of forming a blood vessel cell culturing patterncomprising the cell adhesion portion and the cell adhesion-inhibitingportion in the cell culturing layer, it is not particularly limited. Itmay also be a layer having the cell adhesive properties or a layerhaving the cell adhesion-inhibiting properties. Moreover, in the presentinvention, if it is necessary, the cell culturing layer may be formed onthe base material.

In the present process, if it is a method capable of forming a bloodvessel cell culturing pattern having the cell adhesion portion and thecell adhesion-inhibiting portion, a forming method, is not particularlylimited. For example, it can be made a method in which a mask having thelight shielding portion in a pattern form of the blood vessel formationpattern and a material having the cell adhesion-inhibiting properties iscoated in a pattern form on the cell culturing layer having the celladhesive properties by a printing method and the like; or a method inwhich a layer having the cell adhesion-inhibiting properties is formedon the cell culturing layer having the cell adhesive properties andpatterned in a pattern form of the cell adhesion-inhibiting portion by aphotolithography method or the like. Moreover, it may be a method inwhich a mask having the opening portion in a pattern form of the bloodvessel formation pattern is formed and a material having the celladhesive properties is coated in a pattern form on the cell culturinglayer having the cell adhesion-inhibiting properties to form the celladhesion portion; or a forming method of the cell adhesion portion bypatterning with a photolithography method similar to the description setforth above.

Here, in the present invention, it is preferable that theabove-described cell culturing pattern is formed by irradiating theenergy on the cell culturing layer in which the cell adhesion portionand the above-described cell adhesion-inhibiting portion can be formedby the action of the photocatalyst accompanying with the energyirradiation. In this case, the formation of the cell adhesion portionand the cell adhesion-inhibiting portion can be easily performed withoututilizing chemicals or the like which adversely affect the blood vesselcells and without performing the complex process. It should be notedthat as a forming method of the blood vessel cell culturing pattern bythe action of the photocatalyst accompanying with the energyirradiation, the following six embodiments are listed. Hereinafter, eachof embodiments will be explained.

(1) First Embodiment

First, as a first embodiment, the cell culturing layer is aphotocatalyst-containing cell adhesion layer comprising at leastphotocatalyst, the adhesive properties with cells and a blood vesselcell adhesive material which is decomposed or denatured by the action ofa photocatalyst accompanying with the energy irradiation; and it is acase where for example, the cell adhesion-inhibiting portion is formedby decomposing or denaturing the blood vessel cell adhesive material byirradiating energy using such as a photomask having the light shieldingportion in a pattern form of the blood vessel formation pattern on thephotocatalyst-containing cell adhesion layer.

According to the present embodiment, since the photocatalyst-containingcell adhesion layer contains a photocatalyst and the blood vessel celladhesive material, the area where the energy has been irradiated can bemade the cell adhesion-inhibiting portion which is not adhered to theblood vessel cells by decomposing or denaturing the blood vessel celladhesive material. On the other hand, since the area where the energy isnot irradiated can be made the cell adhesion portion which is excellentin the adhesive properties with the blood vessel cells since the bloodvessel cell adhesive material remains.

Moreover, according to the present embodiment, in the process forculturing blood vessel cell described later, the blood vessel cellsadhered to the cell adhesion-inhibiting portion can be removed by theaction of the photocatalyst by irradiating energy to the celladhesion-inhibiting portion at the time when the blood vessel cell isadhered to the cell adhesion portion, cultured and made into a tissue.Owing to this, it has an advantage that the blood vessels can be formedin a further finely processed pattern.

Hereinafter, a forming method of a photocatalyst-containing celladhesion layer and a cell adhesion-inhibiting portion which are used inthe present embodiment will be explained below.

a. Photocatalyst-Containing Cell Adhesion Layer

First, a photocatalyst-containing cell adhesion layer used in thepresent embodiment will be explained below. A photocatalyst-containingcell adhesion layer used in the present embodiment at least contains aphotocatalyst and the above-described blood vessel cell adhesivematerial, and the photocatalyst-containing layer is a layer in which theblood vessel cell adhesive material is decomposed or denatured by theaction of the photocatalyst accompanying with the energy irradiation tolower the adhesive properties with the cell.

The formation of the photocatalyst-containing cell adhesion layerdescribed above can be performed, for example, by coating, on a basematerial, a coating solution for forming a photocatalyst-containing celladhesion layer containing: a blood vessel cell adhesive material whichis decomposed and denatured by the action of the photocatalystaccompanying with the energy irradiation, and the photocatalyst. Thecoating of the coating solution for formation of thephotocatalyst-containing cell adhesion layer can be performed using ageneral method of coating such as a spin coat method, a spray coatmethod, a dip coat method, a roll coat method, and a bead coat method.

At this time, as the thickness of a film of the photocatalyst-containingcell adhesion layer, usually, it is in the range from about 0.01 μm toabout 1.0 μm, and it is preferably in the range from about 0.1 μm toabout 0.3 μm.

Hereinafter, the respective materials used in thephotocatalyst-containing cell adhesion layer used in the presentembodiment will be explained below.

(i) Blood Vessel Cell Adhesive Material

First, a blood vessel cell adhesive material contained in aphotocatalyst-containing cell adhesion layer of the present embodimentwill be explained below. As for a blood vessel cell adhesive materialcontained in a photocatalyst-containing cell adhesion layer of thepresent embodiment, the kind or the like is not particularly limited aslong as it has the adhesive properties with blood vessel cells and it isdecomposed or denatured by the action of the photocatalyst accompanyingwith the energy irradiation.

The blood vessel cell adhesive material used in the present embodimenthas the adhesive properties with the blood vessels, and such as thosewhich loses the adhesive properties with the blood vessel cell or thosebeing changed to have the cell adhesion-inhibiting properties forinhibiting the adhesion with the blood vessel cell by being decomposedor denatured by the action of the photocatalyst accompanying with theenergy irradiation.

As such materials having the adhesive properties to a blood vessel cell,there are two kinds. One is being materials having the adhesiveproperties to a blood vessel cell owing to physicochemicalcharacteristics and the other being materials having the adhesiveproperties to a blood vessel cell owing to biochemical characteristics.

As physicochemical factors that determine the adhesive properties to ablood vessel cell of the materials having the adhesive properties to ablood vessel cell owing to the physicochemical characteristics, thesurface free energy, the electrostatic interaction and the like can becited. For instance, when the adhesive properties to a blood vessel cellis determined by the surface free energy of the material, the adhesiveproperties between the blood vessel cell and the material becomes goodwhen the material has the surface free energy in a predetermined range.If it deviates from the predetermined range the adhesive propertiesbetween the blood vessel cell and material is deteriorated. As suchchanges of the adhesive properties to a blood vessel cell due to thesurface free energy, experimental results shown in Data, for instance,CMC Publishing Co., Ltd. “Biomaterial no Saisentan”, Yoshito IKADA(editor), p. 109, lower part are known. As materials having the adhesiveproperties to a blood vessel cell owing to such a factor, for instance,hydrophilic polystyrene, and poly (N-isopropyl acrylamide) can be cited.When such a material is used, by the action of the photocatalyst uponirradiation with energy, for instance, a functional group on a surfaceof the material is substituted, decomposed or the like to cause a changein the surface free energy, resulting in one that does not have theadhesive properties to a blood vessel cell or one that has the celladhesion-inhibiting properties.

When the adhesive properties between blood vessel cell and a material isdetermined owing to such as the electrostatic interaction, the adhesiveproperties to a blood vessel cell are determined by such as an amount ofpositive electric charges that the material has. As materials having theadhesive properties to a blood vessel cell owing to such electrostaticinteraction, basic polymers such as polylysine; basic compounds such asaminopropyltriethoxysilane,N-(2-aminoethyl)-3-aminopropyltrimethoxysilane; and condensates and thelike including these can be cited. When such materials are used, by theaction of the photocatalyst upon irradiation with energy, theabove-mentioned materials are decomposed or denatured. Thereby, forinstance, an amount of positive electric charges present on a surfacecan be altered, resulting in one that does not have the adhesiveproperties to a blood vessel cell or one that has the celladhesion-inhibiting properties.

As materials having the adhesive properties to a blood vessel cell owingto the biological characteristics, ones that are good in the adhesiveproperties with particular blood vessel cell or ones that are good inthe adhesive properties with many blood vessel cells can be cited.Specifically, fibronectin, laminin, tenascin, vitronectin, RGD(arginine-glycine-asparagine acid) sequence containing peptide, YIGSR(tyrosine-isoleucine-glycine-serine-arginine) sequence containingpeptide, collagen, atelocollagen, and gelatin can be cited. When suchmaterials are used, by the action of the photocatalyst upon irradiationwith energy, for instance, a structure of the material is partiallydestroyed, or a principal chain is destroyed, resulting in one that doesnot have the adhesive properties to a blood vessel cell or one that hasthe cell adhesion-inhibiting properties.

Such a blood vessel cell adhesive material, though it differs dependingon the kind of the materials and the like, is comprised in thephotocatalyst-containing cell adhesion layer normally in the range of0.01% by weight to 95% by weight, and preferably in the range of 1% byweight to 10% by weight. Thereby, a region that contains the bloodvessel cell adhesive material can be made a region good in the adhesiveproperties to a blood vessel cell.

(ii) Photocatalyst

Next, a photocatalyst contained in a photocatalyst-containing celladhesion layer of the present embodiment will be explained below. As fora photocatalyst used in the present embodiment, it is not particularlylimited as long as it is a photocatalyst that can decompose or denaturethe blood vessel cell adhesive material by the action of thephotocatalyst accompanying with the energy irradiation.

Herein, the action mechanism of the photocatalyst which is representedby titanium oxide described later is not necessarily clear, however, itis considered that a carrier generated by the irradiation of the lightchanges the chemical structure of the organic material by the directreaction with the compound nearby, or by the active oxygen speciesgenerated in the presence of water or oxygen. In the present embodiment,it is considered that this carrier has the influence on the blood vesselcell adhesive material described above.

As the photocatalyst that can be used in the present embodiment,specifically, for instance, titanium dioxide (TiO₂), zinc oxide (ZnO),tin oxide (SnO₂), strontium titanate (SrTiO₃), tungsten oxide (WO₃),bismuth oxide (Bi₂O₃) and iron oxide (Fe₂O₃) that are known asphoto-semiconductors can be cited. These can be used singularly or incombination of at least two kinds.

In the present embodiment, in particular, titanium dioxide, owing to alarge band gap, chemical stability, non-toxicity, and easy availability,can be preferably used. There are two types of titanium dioxide, anatasetype and rutile type, and both can be used in the present embodiment;however, the anatase type titanium dioxide is more preferable. Anexcitation wavelength of the anatase type titanium dioxide is 380 nm orless.

As such anatase type titanium dioxide, for instance, an anatase titaniasol of hydrochloric acid deflocculation type (trade name: STS-02,manufactured by ISHIHARA SANGYO KAISHA, LTD., average particle diameter:7 nm, and trade name: ST-KO1, manufactured by ISHIHARA SANGYO KAISHA,LTD.), and an anatase titania sol of nitric acid deflocculation type(trade name: TA-15, manufactured by NISSAN CHEMICAL INDUSTRIES, LTD.,average particle diameter: 12 nm) can be cited.

The smaller is a particle diameter of the photocatalyst, the better,because a photocatalyst reaction is caused more effectively. It ispreferable to use the photocatalyst with an average particle diameter of50 nm or less, and one having an average particle diameter of 20 nm orless can be particularly preferably used.

The contents of a photocatalyst in a photocatalyst-containing celladhesion layer of the present embodiment can be set in the range from 5to 95% by weight, preferably in the range from 10 to 60% by weight, andmore preferably in the range from 20 to 40% by weight.

It is because this enables a blood vessel cell adhesive material in thearea of the photocatalyst-containing cell adhesion layer where theenergy has been irradiated to be decomposed or denatured.

Herein, as for a photocatalyst used in the present embodiment, it ispreferable that its adhesive properties with blood vessel cells are lowby, for example, having a high hydrophilicity. It is because thisenables the area where the photocatalyst has been exposed by the bloodvessel cell adhesive material being decomposed and so on to be used asan area where the adhesive properties with the blood vessel cells islow.

(iii) The Others

In this embodiment, not only the blood vessel cell adhesive material orthe photocatalyst but also a binder etc. for improving such as strengthor resistance may be contained as necessary in thephotocatalyst-containing cell adhesion layer. In the present embodiment,particularly as the binder, a material that, at least after the energyirradiation, has the cell adhesion-inhibiting properties of inhibitingadhesion to the blood vessel cell is preferably used. This is becausethe adhesion between the blood vessel cell and the celladhesion-inhibiting portion, which is a region irradiated with energy,can thereby be reduced. As such a material, for example, one that hasthe cell adhesion-inhibiting properties prior to the energy irradiationor one that obtains the cell adhesion-inhibiting properties by theaction of the photocatalyst upon irradiation with energy may be used.

In the present embodiment, a material that becomes to have the celladhesion-inhibiting properties, particularly by the action of thephotocatalyst upon irradiation with energy, is preferably used as abinder. Thereby, in a region prior to the energy irradiation, theadhesive properties between the blood vessel cell adhesive material andthe blood vessel cell is not inhibited, and only a region where energyis irradiated can be lowered in the adhesive properties to a bloodvessel cell.

As materials that can be used as such a binder, for instance, ones inwhich a principal skeleton has such a high bond energy, that cannot bedecomposed by the photo-excitation of the photocatalyst, and has anorganic substituent which can be decomposed by an action of thephotocatalyst are preferably used. For instance, (1) organopolysiloxanethat exhibits large strength by hydrolyzing or polycondensating chloro-or alkoxysilane or the like owing to a sol-gel reaction and the like,and (2) organopolysiloxane and the like in which reactive siliconesexcellent in the water repellency or oil repellency are crosslinked canbe cited. For example, those disclosed in JP-A No. 2000-249821 can beused.

When the above-mentioned material is used as the blood vessel celladhesion-inhibiting material, the contact angle thereof with water ispreferably in the range of 15° to 120°, more preferably 20° to 100°before the material is irradiated with energy. According to this, theadhesion of the blood vessel cell adhesive material to the blood vesselcell is not inhibited.

In the case of irradiating this blood vessel cell adhesion-inhibitingmaterial with energy, it is preferred that the contact angle thereofwith water becomes 10° or less. This range makes it possible to renderthe material having a high hydrophilicity and low adhesive properties toa blood vessel cell.

The contact angle with water referred to herein is a result obtained byusing a contact angle measuring device (CA-Z model, manufactured byKyowa Interface Science Co., Ltd.) to measure the contact angle of thematerial with water or a liquid having a contact angle equivalent tothat of water (after 30 seconds from the time when droplets of theliquid are dropped down from its micro syringe), or a value obtainedfrom a graph prepared from the result.

In the present embodiment, a decomposing material or the like thatcauses such as a change in the wettability of a region where energy isirradiated, thereby lowers the adhesive properties to a blood vesselcell or that aides such a change, or other kinds of additive agents maybe contained.

As a decomposing material or an additive agent, for example, a materialor an agent described in JP-A No. 2000-249821 can be used.

In the present embodiment, such a binder and the like can be preferablycomprised in the photocatalyst-containing cell adhesion layer, in therange of 5% by weight to 95% by weight, more preferably 40% by weight to90% by weight, and particularly preferably 60% by weight to 80% byweight.

Moreover, as a base material used in the present embodiment, it is notparticularly limited as long as it is a base material capable of formingthe photocatalyst-containing cell adhesion layer. As the base materialdescribed above, for example, an inorganic material such as a metal, aglass, or silicone and an organic material represented by a plastic canbe used.

Moreover, the flexibility of a base material is appropriately selected.Moreover, the transparency of the base material is appropriatelyselected according to the direction of the irradiation of the energyirradiated for decomposing or denaturing the blood vessel cell adhesivematerial and the like. For example, in the case where the base materialhas the light shielding portion or the like and where the irradiation ofthe energy is performed from the side of the base material, it is madethat the base material has the transparency.

Herein, in the present embodiment, in the case where the base materialhas the transparency for the energy irradiated, the light shieldingportion may be formed in a form for forming the cell adhesion portion,specifically, in a form of the blood vessel forming pattern. It isbecause owing to this, at the time of forming the celladhesion-inhibiting portion by irradiating the energy, the blood vesselcell adhesive material in the photocatalyst-containing cell adhesionlayer only in the targeted area can be decomposed or denatured, withoutusing such as a photomask, by irradiating the energy to the totalsurface from the rear surface side of the base material. As the lightshielding portion described above, a light shielding portion similar tothe light shielding portion generally used can be used, thus, thedetailed explanation is not repeated here.

b. Forming Method of Cell Adhesion-Inhibiting Portion

Next, a forming method of a cell adhesion-inhibiting portion in thepresent embodiment will be explained below. In the present embodiment,for example, as shown in FIGS. 3A and 3B; the blood vessel cell adhesivematerial in the photocatalyst-containing cell adhesion layer 12 in thearea where the energy has been irradiated is decomposed or denatured,the cell adhesion-inhibiting portion 3 which does not have the adhesiveproperties with the blood vessel cells can be formed and it can be madethat the area where the energy has not been irradiated the cell adhesionportion 2 (FIG. 3B), by irradiating the energy 14 on thephotocatalyst-containing cell adhesion layer 12 formed on the basematerial 11 using such as the photomask 13 having the light shieldingportion in a form of the blood vessel formation pattern (FIG. 3A). Atthis time, in the cell adhesion-inhibiting portion, the photocatalystand the decomposed material or the denatured material the blood vesselcell adhesive material are contained.

The energy irradiation (exposure) mentioned in this embodiment is aconcept that includes all energy ray irradiation that can decompose ordenature the blood vessel cell adhesive material by the action of thephotocatalyst upon irradiation with energy, and is not limited to lightirradiation.

Normally, as the light used in such energy irradiation, the wavelengthof the light is set in the range of 400 nm or less, preferably 380 nm orless. This is because, as mentioned above, the photocatalyst that ispreferably used as a photocatalyst is titanium dioxide, and as energythat activates a photocatalyst action by the titanium oxide, the lighthaving the above-mentioned wavelength is preferable.

As a light source that can be used in such energy irradiation, a mercurylamp, metal halide lamp, xenon lamp, excimer lamp and other variouskinds of light sources can be cited.

Other than the method in which pattern irradiation is carried out via aphotomask by using the above-mentioned light source, a method ofcarrying out drawing irradiation in a pattern by using laser such asexcimer or YAG can be applied. Furthermore, as mentioned above, when thebase material has the light-shielding portion in a pattern same as thatof the cell adhesion portion, energy can be irradiated over the entiresurface from the base material side. In this case, there are advantagesin that there are no needs of the photomask and the like and a processof positional alignment and the like are also not necessary.

An amount of irradiation of energy at the energy irradiation is anamount of irradiation necessary for decomposing or denaturing the bloodvessel cell adhesive material by the action of the photocatalyst.

At this time, by energy irradiating a layer containing the photocatalystwhile heating, the sensitivity can be raised; accordingly, it ispreferable in that the blood vessel cell adhesive material can beefficiently decomposed or denatured. Specifically, it is preferable toheat in the range of 30° C. to 80° C.

The energy irradiation that is carried out via a photomask in thisembodiment, when the above-mentioned base material is transparent, maybe carried out from either direction of the base material side or aphotocatalyst-containing cell adhesion layer side. On the other hand,when the base material is opaque, it is necessary to irradiate energyfrom a photocatalyst-containing cell adhesion layer side.

(2) Second Embodiment

Next, as a second embodiment, the cell culturing layer is aphotocatalyst-containing cell adhesion-inhibiting layer containing atleast a photocatalyst, and a blood vessel cell adhesion-inhibitingmaterial having the cell adhesion-inhibiting properties for inhibitingthe adhesion with the blood vessel cell and decomposed or denatured bythe action of the photocatalyst accompanying with the energyirradiation; and it is a case where a cell adhesion portion is formed bydecomposing or denaturing the blood vessel cell adhesion-inhibitingmaterial by irradiating the energy on the photocatalyst-containing celladhesion-inhibiting layer, for example, using a photomask having theopening portion in a pattern form of the blood vessel formation pattern.

In the present embodiment, since the photocatalyst-containing celladhesion-inhibiting layer contains the photocatalyst and the bloodvessel cell adhesion-inhibiting material, the blood vessel celladhesion-inhibiting material can be decomposed or denatured and the areawhere the energy has been irradiated can be made as a cell adhesionportion having the adhesive properties with the blood vessel cells,through the action of the photocatalyst contained in the layer byirradiating the energy in a pattern form of the blood vessel formationpattern on the area where the cell adhesion portion is formed Moreover,at this time, as for the area where the energy is not irradiated, theblood vessel cell adhesion-inhibiting material still remains, and it canbe made as the cell adhesion-inhibiting portion.

Hereinafter, a photocatalyst-containing cell adhesion-inhibiting layerand a base material used in the present embodiment will be explained,and further, a forming method of a cell adhesion portion will beexplained below.

a. Photocatalyst-Containing Cell Adhesion-Inhibiting Layer

First, a photocatalyst-containing cell adhesion-inhibiting layer used inthe present embodiment will be explained below. Aphotocatalyst-containing cell adhesion-inhibiting layer used in thepresent embodiment is a layer containing the photocatalyst and the bloodvessel cell adhesion-inhibiting material, and it is to be a layer havingthe adhesive properties with the blood vessel cells by decomposing ordenaturing the blood vessel cell adhesion-inhibiting material throughthe action of the photocatalyst accompanying with the energyirradiation.

The formation of the above-described photocatalyst-containing celladhesion-inhibiting layer can be performed by, for example, coating onthe base material a coating solution for forming thephotocatalyst-containing cell adhesion-inhibiting layer containing theblood vessel cell adhesion-inhibiting material which is decomposed ordenatured by the action of the photocatalyst accompanying with theenergy irradiation and the photocatalyst. The coating of the coatingsolution for forming the photocatalyst-containing celladhesion-inhibiting layer can be performed by utilizing a general methodof coating such as a spin coat method, a spray coat method, a dip coatmethod, a roll coat method, and a bead coat method

At this time, as the thickness of the film of thephotocatalyst-containing cell adhesion-inhibiting layer, it is usuallyin the range from about 0.01 μm to about 1.0 μm, and particularly in therange form about 0.1 μm to about 0.3 μm.

Hereinafter, a material used in the photocatalyst-containing celladhesion-inhibiting layer will be explained below. Since thephotocatalyst used in the present embodiment can be similar to thephotocatalyst used in the above-described first embodiment, the detailedexplanation is not repeated here.

(i) Blood Vessel Cell Adhesion-Inhibiting Material

First, a blood vessel cell adhesion-inhibiting material contained in thephotocatalyst-containing cell adhesion-inhibiting layer used in thepresent embodiment will be explained below.

As for a blood vessel cell adhesion-inhibiting material used in thepresent embodiment, the kind of it and other properties is notparticularly limited if it is a blood vessel cell adhesion-inhibitingmaterial having the cell adhesion-inhibiting properties for inhibitingthe adhesion with the blood vessel cells and it is decomposed ordenatured by the action of the photocatalyst accompanying with theenergy irradiation.

For the blood vessel cell adhesion-inhibiting material used in thepresent embodiment, those having the above-described celladhesion-inhibiting properties in which the cell adhesion-inhibitingproperties are lost or become to show the cell adhesive properties bybeing decomposed or denatured by the action of the photocatalystaccompanying with the energy irradiation can be used.

As the blood vessel cell adhesion-inhibiting material, a material havinghigh hydration ability can be used as an example. The material havinghigh hydration ability forms a hydration layer wherein water moleculesgather around thereof. Usually, since such a material having highhydration ability has higher adhesion to water molecules than adhesionto the blood vessel cell, the blood vessel cell cannot be adhered to thematerial having high hydration ability. Thus, the layer will have lowadhesive properties to the blood vessel cell. The hydration ability isreferred to as a property of hydrating with water molecules, and highhydration ability is intended to mean that the material is easilyhydrated with water molecules.

As the material having high hydration ability and is used as a bloodvessel cell adhesion-inhibiting material, for example, polyethyleneglycol, amphoteric ionic materials having a betaine structure, orphospholipid-containing materials can be listed. When such materials areused as the blood vessel cell adhesion-inhibiting material, uponirradiated with energy in the below-described energy irradiatingprocess, the blood vessel cell adhesion-inhibiting material isdecomposed or denatured by the action of a photocatalyst so as to removethe hydration layer on the surface, thereby obtaining the material nothaving the cell adhesion-inhibiting properties.

In this embodiment, a surfactant, which is decomposed by the action of aphotocatalyst and has water repellent or oil repellent organicsubstituent, can also be used as the blood vessel celladhesion-inhibiting material. As such surfactant for example, nonionicsurfactants such as: hydrocarbon based such as the respective series ofNIKKOL BL, BC, BO, and BB manufactured by Nikko Chemicals Co., Ltd.; andfluorine based or silicone based such as ZONYL FSN and FSO manufactureby Du Pont Kabushiki Kaisha, Surflon S-141 and 145 manufactured by ASAHIGLASS CO., LTD., Megaface F-141 and 144 manufactured by DAINIPPON INKAND CHEMICALS, Inc., FTERGENT F-200 and F251 manufactured by Neos,UNIDYNE DS-401 and 402 manufactured by DAIKIN INDUSTRIES, Ltd., andFluorad FC-170 and 176 manufactured by 3M can be cited. Also, cationicsurfactants, anionic surfactants and amphoteric surfactants also can beused.

When the photocatalyst-containing cell adhesion-inhibiting layer isformed by using the above material as the blood vessel celladhesion-inhibiting material, the blood vessel cell adhesion-inhibitingmaterial is unevenly distributed on the surface. The water repellency oroil repellency on the surface can thereby be increased, and theinteraction with the blood vessel cell can be decreased to reduceadhesive properties to the blood vessel cell. Upon irradiation of thislayer with energy in the energy irradiating process, the material iseasily decomposed by the action of the photocatalyst to expose thephotocatalyst. Thus, one not having the cell adhesion-inhibitingproperties can be obtained.

In this embodiment, a material, which obtains good adhesive propertiesto a blood vessel cell by the action of the photocatalyst uponirradiation with energy, is particularly preferably used as the bloodvessel cell adhesion-inhibiting material. As such blood vessel celladhesion-inhibiting material, for example, materials having oilrepellency or water repellency can be listed.

When the material having oil repellency or water repellency is used asthe blood vessel cell adhesion-inhibiting material, the interaction suchas hydrophobic interaction between the blood vessel cell and the bloodvessel cell adhesion-inhibiting material is made low by the waterrepellency or oil repellency of the blood vessel celladhesion-inhibiting material, thereby decreasing adhesive properties tothe blood vessel cell.

As the material having water repellency or oil repellency, a material,for example, which has such high bonding energy that the skeletonthereof is not decomposed by the action of the photocatalyst and haswater repellent or oil repellant organic substituent to be decomposed byaction of the photocatalyst, can be listed.

Examples of such a material, which has such high bonding energy that theskeleton thereof is not decomposed by the action of the photocatalystand has water repellent or oil repellant organic substituent to bedecomposed by action of the photocatalyst, include, for example, thematerials used as the binder in the first embodiment, that is, (1) theorganopolysiloxanes exhibiting high strength, obtained by hydrolyzing orpolycondensating chloro- or alkoxysilanes by sol-gel reaction etc., and(2) organopolysiloxanes obtained by crosslinking reactive silicone.

When such material is used as the binder in the first embodiment, thematerial is used as a material having cell adhesion-inhibitingproperties by decomposing or denaturing the side chains of theorganopolysiloxanes, in high ratio, so as to make it super hydrophilicby the action of the photocatalyst upon irradiation with energy.However, in this embodiment, the region irradiated with the energy canhave adhesive properties to a blood vessel cell by irradiating withenergy to such a degree that side chains of the organopolysiloxanes arenot completely decomposed or denatured by the action of thephotocatalyst upon irradiation with energy. Together with theabove-mentioned organopolysiloxanes, a stable organosilicon compound notundergoing any crosslinking reaction, such as dimethylpolysiloxane, canalso be separately mixed.

When the material having water repellency or oil repellency is used asthe blood vessel cell adhesion-inhibiting material, the materialpreferably has a contact angle, with water, of 80° or more, particularlyin the range of 100° to 130°. With this contact angle given, theadhesive properties to a blood vessel cell of thephotocatalyst-containing cell adhesion-inhibiting layer beforeirradiation with energy can be reduced. The upper limit of the angle isthe upper limit of the contact angle, with water, of the blood vesselcell adhesion-inhibiting material on a flat base material. For example,when the contact angle, with water, of the blood vessel celladhesion-inhibiting material on a base material with concavoconvex ismeasured, the upper limit may be about 160° as shown by Ogawa et al. inJapanese Journal of Applied Physics, Part 2, Vol. 32, L614-L615, 1993.

When this blood vessel cell adhesion-inhibiting material is irradiatedwith energy to impart the adhesive properties to the blood vessel cell,the material is preferably irradiated with energy such that the contactangle thereof with water comes to be in the range of 10° to 40°,particularly 15° to 30°. The adhesive properties to the blood vesselcell of the photocatalyst-containing cell adhesion-inhibiting layerafter energy irradiation can thereby be increased. The contact anglewith water can be obtained by the method described above.

The blood vessel cell adhesion-inhibiting material is containedpreferably in the range of 0.01% by weight to 95% by weight,particularly 1% by weight to 10% by weight, in thephotocatalyst-containing cell adhesion-inhibiting layer. The regioncontaining the cell adhesion-inhibiting material can thereby be a regionof low adhesive properties to the blood vessel cell.

The cell adhesion-inhibiting material preferably has surface activity.For example, when drying the photocatalyst-containing celladhesion-inhibiting layer-forming coating solution containing the bloodvessel cell adhesion-inhibiting material after coating thereof, thematerial is distributed highly unevenly on the surface of the coatingfilm, thus giving excellent cell adhesion-inhibiting properties.

(ii) Others

The photocatalyst-containing cell adhesion-inhibiting layer in thisembodiment may contain a binder and the like in accordance with requiredcharacteristics such as coating properties in formation of the layer,strength and resistance of the formed layer. The blood vessel celladhesion-inhibiting material may also function as the binder.

As the binder, for example, a binder having such high bonding energythat its principal skeleton is not decomposed by the action of thephotocatalyst can be used. Specific examples of the binder include suchas polysiloxane not having organic substituents or having organicsubstituents to such a degree that adhesive properties are not adverselyaffected, and such polysiloxane can be obtained by hydrolyzing orpolycondensating such as tetramethoxysilane or tetraethoxysilane.

In this embodiment, the binder is contained preferably in the range of5% by weight to 95% by weight, more preferably 40% by weight to 90% byweight, still more preferably 60% by weight to 80% by weight, in thephotocatalyst-containing cell adhesion-inhibiting layer. Byincorporation of the binder in this range, formation of thephotocatalyst-containing cell adhesion-inhibiting layer can befacilitated and the photocatalyst-containing cell adhesion-inhibitinglayer can be endowed with strength etc., thus allowing it to exhibit itscharacteristics.

In this embodiment, the photocatalyst-containing celladhesion-inhibiting layer preferably contains a blood vessel celladhesive material having adhesive properties to the blood vessel cell,at least after irradiation with energy. By this, in thephotocatalyst-containing cell adhesion-inhibiting layer, adhesiveproperties to the blood vessel cell of the cell adhesion portion, whichis the region irradiated with energy, can be further improved. The bloodvessel cell adhesive material may be a material usable as the binder ormay be a material used separately from the binder. The blood vessel celladhesive material may have good adhesive properties to the blood vesselcell prior to irradiation with energy, or may be endowed with goodadhesive properties to the blood vessel cell by the action of thephotocatalyst upon irradiation with energy.

In this embodiment, as long as the blood vessel cell adhesive materialhave good adhesive properties to a blood vessel cell at least afterbeing irradiated with energy, the adhesive properties to a blood vesselcell can be improved, for example, by biological characteristics or byphysical interaction such as hydrophobic interaction, electrostaticinteraction, hydrogen bonding, and van der Waals force.

In this embodiment, the blood vessel cell adhesive material is containedpreferably in the range of 0.01% by weight to 95% by weight,particularly 1% by weight to 10% by weight, in thephotocatalyst-containing cell adhesion-inhibiting layer. By this, thephotocatalyst-containing cell adhesion-inhibiting layer can furtherimprove the adhesive properties to the blood vessel cell of the celladhesion portion, which is a region irradiated with energy. When thematerial having good adhesive properties to the blood vessel cell priorto irradiation with energy is used as the blood vessel cell adhesivematerial, the material is preferably contained to such a degree as notto inhibit the cell adhesion-inhibiting properties of the blood vesselcell adhesion-inhibiting material in the region not irradiated withenergy, that is, the region serving as the cell adhesion-inhibitingportion.

Moreover, as for the base material used in the present embodiment, it isnot particularly limited if it is a base material which is capable offorming the above-described photocatalyst-containing celladhesion-inhibiting layer, and it can be made the base material wexplained in the first embodiment.

Herein, in the present embodiment, in the case where the base materialhas the transparency for the energy to be irradiated, the lightshielding portion may be formed in the area where it is made the celladhesion-inhibiting portion. It is because owing to this, at the timewhen it is made cell adhesion portion by irradiating the energy on thephotocatalyst-containing cell adhesion-inhibiting layer, it is notnecessary to use a photomask or the like, the cell adhesion portion canbe easily formed by irradiating the energy on the whole surface from therear surface of the base material.

Herein, as for the kind of a base material, a forming method of theabove-described light shielding portion and the kind thereof used in thepresent embodiment, since it is similar to these explained in the firstembodiment, the detailed explanation is not repeated here.

b. Forming Method of Cell Adhesion Portion

Next, a forming method of a cell adhesion portion will be explainedbelow. In the present embodiment, the energy is irradiated on thephotocatalyst-containing cell adhesion-inhibiting layer, for example,using a photomask having the opening portion in a form of the bloodvessel formation pattern. It is because owing to this, the celladhesion-inhibiting material in the area where the energy has beenirradiated can be decomposed or denatured t to make the cell adhesionportion having the adhesive properties with the blood vessel cell. Atthe time, a photocatalyst, the decomposed material or the denaturedmaterial of the blood vessel cell adhesion-inhibiting material and thelike are contained in the cell adhesion portion. On the other hand, theblood vessel cell adhesion-inhibiting material which is the area wherethe energy is not irradiated remains, and it can be made celladhesion-inhibiting portion not having the adhesive properties with theblood vessel cell.

As for the above-described method of irradiating the energy or the like,the detailed explanation is not repeated here since it is similar tothese explained in the first embodiment described above.

(3) Third Embodiment

Next, as a third embodiment; the cell culturing layer is a blood vesselcell adhesion layer having the adhesive properties with the blood vesselcell and containing a blood vessel cell adhesive material decomposed ordenatured by the action of the photocatalyst accompanying with theenergy irradiation, formed on the photocatalyst-containing layercontaining at least a photocatalyst; and it is a case where a celladhesion-inhibiting portion is formed on the blood vessel cell adhesionlayer by decomposing or denaturing the blood vessel cell adhesivematerial by irradiating the energy using for example a photomask havingthe light shielding portion in a pattern form of the blood vesselformation pattern.

In the present embodiment, since the blood vessel cell adhesion layer isformed on the photocatalyst-containing layer by the energy irradiation,the blood vessel cell adhesive material in the blood vessel celladhesion layer is decomposed or denatured by the action of the adjoinedphotocatalyst in the photocatalyst-containing layer to enable to formthe cell adhesion-inhibiting portion whose adhesive properties with theblood vessel cells in the area has been lowered. At this time, in thecase where for example, the blood vessel cell adhesive material isdecomposed by the action of the photocatalyst accompanying with theenergy irradiation, the small amount of the blood vessel cell adhesivematerial is contained in the cell adhesion-inhibiting portion thedecomposed material of the blood vessel cell adhesive material or thelike is contained, or the photocatalyst-containing layer is exposed andso on by completely decomposing and removing the blood vessel celladhesion layer. Moreover, in the case where the blood vessel celladhesive material is denatured by the action of the photocatalystaccompanying with the energy irradiation, the denatured material or thelike is contained in the cell adhesion-inhibiting portion.

Moreover, according to the present embodiment, in the process forculturing a blood vessel cell described later, at the time when theblood vessel cells are attached to the cell adhesion portion, culturedand made into a tissue, the blood vessel cells which have been attachedto the cell adhesion-inhibiting portion can be removed and so on by theaction of the photocatalyst by irradiating the energy to the celladhesion-inhibiting portion. Owing to this, it has also an advantagethat blood vessels in a further finely processed pattern can be formed.

Hereinafter, the respective constitutions of the present embodiment willbe explained below. The base material and the forming method of a celladhesion-inhibiting portion used in the present embodiment are similarto the first embodiment, and the explanation is not repeated here.

a. Blood Vessel Cell Adhesion Layer

First, a blood vessel cell adhesion layer used in the present embodimentwill be explained below. The blood vessel cell adhesion layer used inthe present embodiment is a layer having a blood vessel cell adhesivematerial having at least the adhesive properties with the cell andlayers generally used as a layer having the adhesive properties with theblood vessel cells can be used.

As a specific blood vessel cell adhesive material, since a cell adhesivematerial similar to the blood vessel cell adhesive material used in thephotocatalyst-containing cell adhesion layer explained in the firstembodiment can be used, the detailed explanation is not repeated here.Moreover, it is preferable that a material having the celladhesion-inhibiting properties explained in the photocatalyst-containingcell adhesion layer which has been explained in the first embodiment isalso contained in the blood vessel cell adhesion layer of thisembodiment. Owing to this, it becomes possible that the adhesiveproperties with the blood vessel cell of the cell adhesion-inhibitingportion which is the energy-irradiated area is lowered.

Moreover, as for the formation of the blood vessel cell adhesion layer,it can performed by coating a coating solution for forming a bloodvessel cell adhesion layer containing the blood vessel cell adhesivematerial by a general coating method. Since it can be made similar to aforming method of the photocatalyst-containing cell adhesion layer ofthe first embodiment, the explanation is repeated here. It should benoted that in the case where a blood vessel cell adhesive material, suchas protein, comparatively expensive, there may be a case where theabsorption method is applied to the formation of the blood vessel celladhesion layer.

The thickness of the film of the blood vessel cell adhesion layer couldbe made in the range from about 0.01 μm to about 1.0 μm, andparticularly in the range from about 0.01 μm to about 0.3 μm.

b. Photocatalyst-Containing Layer

Next, a photocatalyst-containing layer used in the present embodimentwill be explained below. As for a photocatalyst-containing layer used inthe present embodiment, it is not particularly limited as long as it isa layer containing at least photocatalyst. For example, it may be alayer comprising only photocatalyst, or a layer containing the othercomponent such as binder.

As a photocatalyst used in the present embodiment, it is preferable thatit can be made similar to the photocatalyst used in thephotocatalyst-containing cell adhesion layer in the first embodiment andparticularly titanium oxide is used also in the present embodiment.

Here, in the case where a photocatalyst-containing layer comprising onlya photocatalyst has been used, the efficiency concerning with thedecomposition or the denaturation of the blood vessel cell adhesivematerial in the blood vessel cell adhesion layer is enhanced, and it hasthe advantages from the viewpoint of the cost such as by shortening thetreatment time. On the other hand, in the case where aphotocatalyst-containing layer comprising a photocatalyst and a binderhas been used, it has the advantage that the formation of thephotocatalyst-containing layer is easy.

An example of the forming method for the photocatalyst-containing layermade only of a photocatalyst may be a vacuum film-forming method such assputtering, CVD or vacuum vapor deposition. The formation of thephotocatalyst-containing layer by the vacuum film-forming method makesit possible to render the layer a homogeneous photocatalyst-containinglayer made only of a photocatalyst. Thereby, the blood vessel celladhesive material can be decomposed or denatured homogeneously. At thesame time, since the layer is made only of a photocatalyst, the bloodvessel cell adhesive material can be decomposed or denatured moreeffectively, as compared to the case of using also a binder.

Another example of the method for forming the photocatalyst-containinglayer made only of a photocatalyst, is the following method: forexample, in the case that the photocatalyst is titanium dioxide,amorphous titania is formed on the base material, and then, calcinatingso as to phase-change the titania to crystalline titania. The amorphoustitania used in this case can be obtained, for example, by hydrolysis ordehydration condensation of an inorganic salt of titanium, such astitanium tetrachloride or titanium sulfate, or hydrolysis or dehydrationcondensation of an organic titanium compound, such astetraethoxytitanium, tetraisopropoxytitanium, tetra-n-propoxytitanium,tetrabutoxytitanium or tetramethoxytitanium, in the presence of an acid.Next, the resultant is calcinated at 400° C. to 500° C. so as to bedenatured to anatase type titania, and calcinated at 600° C. to 700° C.so as to be denatured to rutile type titania.

In the case of using a binder, the binder preferably having a highbonding energy, wherein its principal skeleton is not decomposed byphotoexcitation of the photocatalyst is used. Examples of such a binderinclude the organopolysiloxanes described in the above-mentioned item“Blood vessel cell adhesion layer”.

In the case of using such organopolysiloxanes as the binder, thephotocatalyst-containing layer can be formed by dispersing aphotocatalyst, the organopolysiloxane as the binder, and optionaladditives if needed into a solvent to prepare a coating solution, andcoating this coating solution onto the base material. The used solventis preferably an alcoholic based organic solvent such as ethanol orisopropanol. The coating can be performed by a known coating method suchas spin coating, spray coating, dip coating, roll coating, or beadcoating. When the coating solution contains an ultraviolet curablecomponent as the binder, the photocatalyst-containing layer can beformed by curing the coating solution through the irradiation ofultraviolet rays.

As the binder, an amorphous silica precursor can be used. This amorphoussilica precursor is preferably a silicon compound represented by thegeneral formula SiX₄, wherein X being halogen, methoxy group, ethoxygroup, acetyl group or the like; silanol which is a hydrolyzate thereof;or polysiloxane having an average molecular weight of 3000 or less.

Specific examples thereof include such as tetraethoxysilane,tetraisopropoxysilane, tetra-n-propoxysilane, tetrabutoxysilane, andtetramethoxysilane. In this case, the photocatalyst-containing layer canbe formed by dispersing the amorphous silica precursor and particles ofa photocatalyst homogeneously into a non-aqueous solvent, hydrolyzingwith water content in the air to form a silanol onto a transparent basematerial, and then subjecting to dehydration polycondensation at roomtemperature. When the dehydration polycondensation of the silanol isperformed at 100° C. or higher, the polymerization degree of the silanolincreases so that the strength of the film surface can be improved. Asingle kind, or two or more kinds of this binding agent may be used.

The content of the photocatalyst in the photocatalyst-containing layercan be set in the range of 5 to 60% by weight, preferably in the rangeof 20 to 40% by weight. The thickness of the photocatalyst-containinglayer is preferably in the range of 0.05 to 10 μm.

Besides the above-mentioned photocatalyst and binder, the surfactant andso on used in the above-mentioned blood vessel cell adhesion layer canbe incorporated into the photocatalyst-containing layer.

Here, in the present embodiment, as for the photocatalyst-containinglayer, it is preferable that on the surface, the adhesive propertieswith the cells is low because the surface is hydrophilic and the like.Owing to this, in the case where the blood vessel cell adhesion layer isdecomposed and so on, and the photocatalyst-containing layer is exposed,the area can be made an area where the adhesive properties with thecells is low.

Moreover, in the present embodiment, the light shielding portion may beformed on the photocatalyst-containing layer in a pattern of forming thecell adhesion portion, specifically, in a pattern form of the bloodvessel formation pattern. It is because owing to this, in the case wherethe energy has been irradiated on the whole surface of the blood vesselcell adhesion layer, the photocatalyst on the area where the lightshielding portion has been formed is not excited, the blood vessel celladhesive material contained in the blood vessel cell adhesion layerother than the area where the light shielding portion has been formedcan be decomposed or denatured. Moreover, in this case, since thephotocatalyst in the area where the light shielding portion has beenformed is not excited, it has an advantage that the direction of theenergy being irradiated is not particularly limited.

As the above-described light shielding portion, since it is possiblethat a light shielding portion similar to the light shielding portionexplained in the first embodiment is used, the detailed explanation isnot repeated here.

(4) Fourth Embodiment

Next, as a fourth embodiment, the cell culturing layer is a blood vesselcell adhesion-inhibiting layer containing a blood vessel celladhesion-inhibiting material having the cell adhesion-inhibitingproperties for inhibiting the adhesion to the cell and decomposed ordenatured by the action of the photocatalyst accompanying with theenergy irradiation; and it is formed on the photocatalyst-containinglayer of the blood vessels containing at least a photocatalyst, and itis the case where the blood vessel cell adhesion-inhibiting material isdecomposed or denatured by irradiating the energy on the blood vesselcell adhesion-inhibiting layer using a photomask and the like having theopening portion, for example, in a pattern form of the blood vesselformation pattern to form the cell adhesion portion.

In the present embodiment, since blood vessel the celladhesion-inhibiting layer has been formed on thephotocatalyst-containing layer, the photocatalyst contained in thephotocatalyst-containing layer is excited, the blood vessel celladhesion-inhibiting material in the blood vessel celladhesion-inhibiting layer can be decomposed or denatured, and the celladhesion portion can be formed by irradiating the energy in a patternform of the blood vessel formation pattern on the blood vessel celladhesion-inhibiting layer. Moreover, at this time, the area where theenergy is not irradiated and the cell adhesion-inhibiting materialremains can be made the cell adhesion-inhibiting portion.

Here, the fact that the blood vessel cell adhesion-inhibiting materialis decomposed or denatured means that the blood vessel celladhesion-inhibiting material is not contained, or the less amount of theblood vessel cell adhesion-inhibiting material is contained when it iscompared to the amount of the blood vessel cell adhesion-inhibitingmaterial contained in the cell adhesion-inhibiting portion. For example,in the case where the blood vessel cell adhesion-inhibiting material isdecomposed by the action of the photocatalyst accompanying with theenergy irradiation, it means that the small amount of the blood vesselcell adhesion-inhibiting material is contained in the cell adhesionportion, the decomposed material of the blood vessel celladhesion-inhibiting material or the like is contained, or the bloodvessel cell adhesion-inhibiting material is completely decomposed andthe photocatalyst-containing layer is exposed. Moreover, in the casewhere the blood vessel cell adhesion-inhibiting material is denatured bythe action of the photocatalyst accompanying with the energyirradiation, the denatured material or the like is contained in the celladhesion portion. In the present embodiment, it is preferable that thecell adhesion material having the adhesive properties with the bloodvessel cell is contained in the above-described cell adhesion portion atleast after the energy has been irradiated. It is because owing to this,that the adhesive properties with the blood vessel cell of the celladhesion portion can be made higher, and it becomes possible that theblood vessel cell is adhered to only the cell adhesion portion in ahighly fine process.

Hereinafter, a blood vessel cell adhesion-inhibiting layer used in thepresent embodiment will be explained below. As for thephotocatalyst-containing layer used in the present embodiment, aphotocatalyst-containing layer similar to the photocatalyst-containinglayer explained in the third embodiment can be used; and as for the basematerial and the forming method of a cell adhesion portion used in thepresent embodiment, these similar to the second embodiment can be used.Therefore, the explanation is not repeated here.

a. Blood Vessel Cell Adhesion-Inhibiting Layer

As for a blood vessel cell adhesion-inhibiting layer used in the presentembodiment, if it is formed on the photocatalyst-containing layer, hasthe cell adhesion-inhibiting properties for inhibiting the adhesion tothe blood vessel cell, and contains the blood vessel celladhesion-inhibiting material decomposed or denatured by the action ofthe photocatalyst accompanying with the energy irradiation, it is notparticularly limited.

In the present embodiment, if the above-described layer can be formed,as for a forming method or the like is not particularly limited. It canbe, for example, formed by coating a coating solution for forming theblood vessel cell adhesion-inhibiting layer containing the blood vesselcell adhesion-inhibiting material on the photocatalyst-containing layerby a general method of coating. Moreover, the thickness of the film ofthe blood vessel cell adhesion-inhibiting layer can be made that it isusually in the range from about 0.01 μm to about 1.0 μm, andparticularly in the range from about 0.1 μm to about 0.3 μm.

Here, as a specific blood vessel cell adhesion-inhibiting material usedfor the blood vessel cell adhesion-inhibiting layer formed in thepresent embodiment, since a cell adhesion-inhibiting material similar tothe blood vessel cell adhesion-inhibiting material used for thephotocatalyst-containing cell adhesion-inhibiting layer explained in thefirst embodiment can be used, the detailed explanation is not repeatedhere. Moreover, it is preferable that a material having the celladhesive properties explained in the photocatalyst-containing celladhesion-inhibiting layer used in the second embodiment is containedalso in the blood vessel cell adhesion-inhibiting layer of the presentembodiment. It is because that owing to this, the adhesive propertieswith the cells of the cell adhesion portion which is the energyirradiated area can be made higher.

(5) Fifth Embodiment

Moreover, as a fifth embodiment, the cell culturing layer is a bloodvessel cell adhesion layer containing a blood vessel cell adhesivematerial having the adhesive properties with the blood vessel cell anddecomposed or denatured by the action of the photocatalyst accompanyingwith the energy irradiation in which; and it is a case where the bloodvessel cell adhesion layer and the photocatalyst-containing layercontaining a photocatalyst are disposed to oppose to each other and theenergy is irradiated using, for example, a photomask having the lightshielding portion in a pattern form of the blood vessel formationpattern to form the cell adhesion-inhibiting portion is formed bydecomposing or denaturing the blood vessel cell adhesive material.

In the present embodiment, the blood vessel cell adhesive material inthe blood vessel cell adhesion layer is decomposed or denatured toenable to form the cell adhesion-inhibiting portion by disposing theblood vessel cell adhesion layer and the photocatalyst-containing layeropposed each other, irradiating the energy in a pattern form for formingthe cell adhesion-inhibiting portion, and by the action of thephotocatalyst in the photocatalyst-containing layer.

Moreover, according to the present embodiment, in the blood vessel cellculturing process described later, at the time when the blood vesselcells are adhered to, cultured on the cell adhesion portion and madeinto a tissue, the blood vessel cells adhered to the celladhesion-inhibiting portion can be removed and so on by the action ofthe photocatalyst by irradiating the energy on the celladhesion-inhibiting portion. Owing to this, it has an advantage that theblood vessels can be formed in a further finely processed pattern.

Hereinafter, a photocatalyst-containing layer side substrate, and aforming method of a cell adhesion-inhibiting portion using thephotocatalyst-containing layer side substrate used in the presentembodiment will be explained below. As for the blood vessel celladhesion layer used in the present embodiment, it is similar to thethird embodiment, and the explanation is not repeated here.

a. Photocatalyst-Containing Layer Side Substrate

First, the photocatalyst-containing layer side substrate, comprising aphotocatalyst-containing layer containing a photocatalyst, used in thisembodiment is described. The photocatalyst-containing layer sidesubstrate used in this embodiment usually comprises aphotocatalyst-containing layer containing a photocatalyst and generallycomprises a base body and a photocatalyst-containing layer formed on thebase body. This photocatalyst-containing layer side substrate may alsohave, for example, photocatalyst-containing layer side light-shieldingportion formed in a pattern form or a primer layer. The following willdescribe each of the constituents of the photocatalyst-containing layerside substrate used in this embodiment.

(i) Photocatalyst-Containing Layer

First, the photocatalyst-containing layer used in thephotocatalyst-containing layer side substrate is described. Thephotocatalyst-containing layer used in this embodiment is notparticularly limited insofar as the layer is constituted such that thephotocatalyst in the photocatalyst-containing layer can cause thedecomposition or denaturation of the blood vessel cell adhesive materialin the adjacent blood vessel cell adhesion layer. Thephotocatalyst-containing layer may be composed of a photocatalyst and abinder or may be made of a photocatalyst only. The property of thesurface thereof may be lyophilic or repellent to liquid.

Moreover, a photocatalyst-containing layer used in the presentembodiment may be formed on the whole surface of the base body, or itmay be formed on the pattern. It is because that: by forming thephotocatalyst-containing layer in a pattern form, the patternirradiation using a photomask or the like is not needed at the time whenthe energy is irradiated to form the cell adhesion-inhibiting portion;and the cell adhesion-inhibiting portion in which the blood vessel celladhesive material contained in the blood vessel cell adhesion layer hasbeen decomposed or denatured can be formed by irradiating the energy onthe whole surface. The method of patterning the photocatalyst-containinglayer is not particularly limited, and the patterning can be performedwith a method such as a photolithography.

Moreover, since only the portion on the blood vessel cell adhesion layerfacing to the photocatalyst-containing layer of the blood vessel celladhesive material is actually decomposed or denatured if the energy isirradiated on the portion where the photocatalyst-containing layer andthe blood vessel cell adhesion layer face each other, the energy may beirradiated from every direction. Further, it has an advantage that theirradiated energy is not particularly limited to parallel one such asparallel light.

Here, as for the photocatalyst-containing layer used in the presentembodiment, it is possible that a photocatalyst-containing layer similarto the photocatalyst-containing layer explained in the third embodimentis used, thus the detailed explanation is not repeated here.

(ii) Base Body

The following will describe the base body used in thephotocatalyst-containing layer side substrate. Usually, thephotocatalyst-containing layer side substrate comprises at least a basebody and a photocatalyst-containing layer formed on the base body. Inthis case, the material which constitutes the base body to be used isappropriately selected depending on the direction of energy irradiationwhich will be detailed later, necessity of the resulting pattern-formingbody to be transparency, or other factors.

The base body used in this embodiment may be a member havingflexibility, such as a resin film, or may be a member having noflexibility, such as a glass substrate. This is appropriately selecteddepending on the method of the energy irradiation.

An anchor layer may be formed on the base body in order to improve theadhesion between the surface of the base body and thephotocatalyst-containing layer. The anchor layer may be made of, forexample, a silane based or titanium based coupling agent.

(iii) Photocatalyst-Containing Layer Side Light-Shielding Portion

The photocatalyst-containing layer side substrate used in thisembodiment may be a photocatalyst-containing layer side substrate onwhich photocatalyst-containing layer side light-shielding portion isformed in a pattern of blood vessel forming pattern. When thephotocatalyst-containing layer side substrate havingphotocatalyst-containing layer side light-shielding portion is used inthis way, at the time of irradiating energy, it is not necessary to useany photomask or to carry out drawing irradiation with a laser light.Since alignment of the photomask and the photocatalyst-containing layerside substrate is not necessary, process can be made simple. Further,since expensive device for drawing irradiation is also not necessary, itis advantageous in costs.

The photocatalyst-containing layer side light shielding portion of thephotocatalyst-containing layer side may be formed between the base bodyand the photocatalyst-containing layer, or may be formed on thephotocatalyst-containing layer. Moreover, it may be formed on thesurface side of the base body contrary to the side where thephotocatalyst-containing layer is formed.

A forming method of the photocatalyst-containing layer side lightshielding portion is not particularly limited, since it is appropriatelyselected and used corresponding to the property of the forming surfaceof the photocatalyst-containing layer side light shielding portion andthe shielding property against the energy which is required, and it canbe made similar to the light shielding portion provided on the basematerial explained in the first embodiment, the detailed explanation isnot repeated here. Moreover, in the case where thephotocatalyst-containing layer side light shielding portion is formedbetween the base body and the photocatalyst-containing layer, a primerlayer may be formed between the photocatalyst-containing layer sidelight shielding portion and the photocatalyst-containing layer. As theprimer layer, for example, a primer layer described in JP-A No.2002-173205 can be used.

b. Forming Method of Cell Adhesion-Inhibiting Portion

Next, a forming method of a cell adhesion-inhibiting portion in thepresent embodiment will be explained below. In the present embodiment,for example, as shown in FIGS. 4A and 4B, the blood vessel cell adhesionlayer 15 formed on the base material 11 and the photocatalyst-containinglayer 22 of the photocatalyst-containing layer side substrate 21 aredisposed at a predetermined interval, and the energy 14 is irradiatedfrom a predetermined direction using such as the photomask 13 (FIG. 4A).Owing to this, the blood vessel cell adhesive material in the area wherethe energy irradiated is decomposed or denatured and the celladhesion-inhibiting portion 3 not having the adhesive properties withthe blood vessel cell is formed in the cell adhesion portion 2 (FIG.4B). At this time, as for the cell adhesion-inhibiting portion, forexample, in the case where the blood vessel cell adhesive material isdecomposed by the action of the photocatalyst accompanying with theenergy irradiation, the small amount of the blood vessel cell adhesivematerial is contained in the cell adhesion-inhibiting portion, thedecomposed material of the blood vessel cell adhesive material and thelike is contained, or the blood vessel cell adhesion layer is completelydecomposed and removed to expose the base material. Moreover, in thecase where the blood vessel cell adhesive material is denatured by theaction of the photocatalyst accompanying with the energy irradiation,the denatured material and the like is contained in the celladhesion-inhibiting portion.

The above-mentioned wording “disposing” means that the layers aredisposed in the state that the action of the photocatalyst cansubstantially work to the surface of the blood vessel cell adhesionlayer, and include not only the state that the two layers actuallycontact each other, but also the state that the photocatalyst-containinglayer and the blood vessel cell adhesion layer are disposed at apredetermined interval. The dimension of the interval is preferably 200μm or less.

In this embodiment, the dimension of the interval is more preferably inthe range of 0.2 μm to 10 μm, even more preferably in the range of 1 μmto 5 μm, since the precision of the pattern to be obtained becomes verygood and further the sensitivity of the photocatalyst becomes high so asto make good efficiency of the decomposition or denaturation of theblood vessel cell adhesive material in the blood vessel cell adhesionlayer. This range of the interval dimension is particularly effectivefor the blood vessel cell adhesion layer which is small in area, whereinthe interval dimension can be controlled with a high precision.

Meanwhile, in the case of treating the blood vessel cell adhesion layerhaving large area, for example, 300 mm×300 mm or more in size, it isvery difficult to make a fine interval as described above between thephotocatalyst-containing layer side substrate and the blood vessel celladhesion layer without contacting each other. Accordingly, when theblood vessel cell adhesion layer has a relatively large area, theinterval dimension is preferably in the range of 10 to 100 μm, morepreferably in the range of 50 to 75 μm. By setting the intervaldimension in the above range, the following problems will not occurthat: deterioration of patterning precision, such as blurring of thepattern; or the sensitivity of the photocatalyst deteriorates so thatthe efficiency of decomposing or denaturing the blood vessel celladhesive material is also deteriorated. Further, there is anadvantageous effect that the blood vessel cell adhesive material is notunevenly decomposed or denatured.

When energy is irradiated onto the blood vessel cell adhesion layerhaving a relatively large area as described above, the dimension of theinterval, in a unit for positioning the photocatalyst-containing layerside substrate and the blood vessel cell adhesion layer inside theenergy irradiating device, is preferably set in the range of 10 μm to200 μm, more preferably in the range of 25 μm to 75 μm. The setting ofthe interval dimension value into this range makes it possible todispose the photocatalyst-containing layer side substrate and the bloodvessel cell adhesion layer without causing a large deterioration inpatterning precision or of sensitivity in the photocatalyst, and withoutbringing the substrate and the layer into contact with each other.

When the photocatalyst-containing layer and the surface of the bloodvessel cell adhesion layer are disposed at a predetermined interval,active oxygen species generated from oxygen and water by action of thephotocatalyst can easily be released. In other words, if the intervalbetween the photocatalyst-containing layer and the blood vessel celladhesion layer is made narrower than the above-mentioned range, theactive oxygen species are not easily released, so as to make the ratefor decomposing or denaturing the blood vessel cell adhesive materialunfavorably small. If the two layers are arranged at an interval largerthan the above-mentioned range, the generated active oxygen species donot reach the blood vessel cell adhesion layer easily. In this casealso, the rate for decomposing or denaturing the blood vessel celladhesive material may become unfavorably small.

The method for disposing the photocatalyst-containing layer and theblood vessel cell adhesion layer to make such a very small intervalevenly therebetween is, for example, a method of using spacers. The useof the spacers in this way makes it possible to make an even interval.At the same time, the action of the photocatalyst does not work onto thesurface of the blood vessel cell adhesion layer in the regions which thespacers contact. Therefore, when the spacers are rendered to have apattern similar to that of the cell adhesion portions, the blood vesselcell adhesive material only inside regions where no spacers are formedcan be decomposed or denatured so that highly precise celladhesion-inhibiting portions can be formed. The use of the spacers alsomakes it possible that the active oxygen species generated by action ofthe photocatalyst reach the surface of the blood vessel cell adhesionlayer, without diffusing, at a high concentration. Accordingly, highlyprecise cell adhesion-inhibiting portion can be effectively formed.

In this embodiment, it is sufficient that such a disposed state of thephotocatalyst-containing layer side substrate is maintained only duringthe irradiation of energy.

The energy irradiation (exposure) mentioned in this embodiment is aconcept that includes all energy ray irradiation that can decompose ordenature the blood vessel cell adhesive material by the action of thephotocatalyst upon irradiation with energy, and is not limited to lightirradiation.

The kind and other properties of the energy irradiated are the same asthose explained in the first embodiment, thus the detailed explanationthereof is omitted here.

The energy irradiation that is carried out via a photomask in thisembodiment, when the above-mentioned base material is transparent, maybe carried out from either direction of the base material side or aphotocatalyst-containing layer side substrate. On the other hand, whenthe base material is opaque, it is necessary to irradiate energy from aphotocatalyst-containing layer side substrate.

(6) Sixth Embodiment

Still further, as a six embodiment, the cell culturing layer is a bloodvessel cell adhesion-inhibiting layer having the adhesion-inhibitingproperties which inhibits adhesion to the blood vessel cell containing ablood vessel cell adhesive-inhibiting material decomposed or denaturedby the action of the photocatalyst accompanying with the energyirradiation; and it is a case where for example, the cell adhesionportion is formed by decomposing or denaturing the blood vessel celladhesion-inhibiting material by disposing the blood vessel cell adhesionlayer and the photocatalyst-containing layer containing a photocatalystopposed to each other and by irradiating the energy using such as aphotomask having the light shielding portion in a pattern form of theblood vessel formation pattern.

In the present embodiment, since the blood vessel celladhesion-inhibiting material decomposed or denatured by the action ofthe photocatalyst accompanying with the energy irradiation is containedin the blood vessel cell adhesion-inhibiting layer, the blood vesselcell adhesion-inhibiting material in the blood vessel celladhesion-inhibiting layer is decomposed or denatured and the celladhesion portion having the adhesive properties with the blood vesselcell can be formed by disposing the blood vessel celladhesion-inhibiting layer and the photocatalyst-containing layer opposedto each other and by irradiating the energy in a pattern form of theblood vessel formation pattern by means of the action of thephotocatalyst in the photocatalyst-containing layer. At this time,concerning with the area where the energy is not irradiated, since theblood vessel cell adhesion-inhibiting material remains, it can be madethe area not having the adhesive properties with the blood vessel cell,and it can be used as a cell adhesion-inhibiting portion.

Here, the fact that the blood vessel cell adhesion-inhibiting materialis decomposed or denatured means that the blood vessel celladhesion-inhibiting material is not contained, or the small amount ofthe blood vessel cell adhesion-inhibiting material is contained when itis compared to the amount of the blood vessel cell adhesion-inhibitingmaterial contained in the cell adhesion-inhibiting portion. For example,in the case where the blood vessel cell adhesion-inhibiting material isdecomposed by the action of the photocatalyst accompanying with theenergy irradiation, the small amount of the blood vessel celladhesion-inhibiting material is contained, the decomposed material ofthe blood vessel cell adhesion-inhibiting material or the like iscontained, or the blood vessel cell adhesion-inhibiting material iscompletely decomposed and the photocatalyst-containing layer is exposed.Moreover, in the case where the blood vessel cell adhesion-inhibitingmaterial is denatured by the action of the photocatalyst accompanyingwith the energy irradiation, the denatured material or the like iscontained in the cell adhesion portion. In the present embodiment, it ispreferable that the cell adhesion material having the adhesiveproperties with the blood vessel cell is contained in the cell adhesionportion at least after the energy has been irradiated. It is becauseowing to this, that the adhesive properties with the blood vessel cellof the cell adhesion portion can be made higher, and it becomes possiblethat the blood vessel cell is adhered to only the cell adhesion portionin a highly fine process.

The blood vessel cell adhesion-inhibiting layer used in the presentembodiment is similar to the blood vessel cell adhesion-inhibiting layerexplained in the fourth embodiment; and the photocatalyst-containinglayer side substrate, the disposition, a method of irradiating theenergy and the like are similar to those explained in the fifthembodiment. Accordingly, the detailed explanations are not repeatedhere.

4. Blood Vessel Cell Culturing Process

Next, the process for culturing a blood vessel cell in the presentinvention will be explained below. The process for culturing a bloodvessel cell in the present invention is a process in which the bloodvessel cells are adhered to the above-described cell adhesion portion ina pattern of the of blood vessel cell culturing pattern, cultured andmade into a tissue. According to the present process, the blood vesselscan be formed by adhering the blood vessel cells to the cell adhesionportion and making into the tissue.

The blood vessel cells used in the present process are blood vesselcells which are cultured and organize the blood vessels, and these meansvascular endothelial cells, pericyte, smooth muscle cells, vascularendothelial precursor cell, smooth muscle precursor cell obtained fromthe respective living organisms, particularly from human and animals,and particularly, vascular endothelial cells and the like can be used.Plural kinds of cells can be co-cultured such as co-culture of vascularendothelial cells and pericytes or co-culture of vascular endothelialcells and smooth muscle cells.

Moreover, as a method of adhering the blood vessel cells to the celladhesion portion, if it is a method of being capable of adhering theblood vessel cells only to the cell adhesion portion in a pattern of theblood vessel cell culturing pattern having the cell adhesion portion andthe cell adhesion-inhibiting portion, it is not particularly limited.For example, it may be a method of adhering the blood vessel cells by anink jet printer or a manipulator, however, the following method isgenerally used: a method in which after the blood vessel cells wereadhered to the cell adhesion portion by disseminating the blood vesselcell suspension, the blood vessel cells on the cell adhesion-inhibitingportion which has been unnecessary is washed with phosphate buffer, andthe blood vessel cells are removed. As the above-described method, forexample, the method described in the reference document “Spatialdistribution of mammalian cells dictated by material surface chemistry”,Kevin E. Healy, et al, Biotech. Bioeng. (1994), p. 792 can be used.

Moreover, in the present invention, blood vessels can be formed by makethe blood vessel cells in contact with the anchorage material forpromoting to make the blood vessel cells into the blood vessels such asMatrigel as it is in a form of the blood vessels adhered to the bloodvessel cell adhesion portion. Still further, it is also possible thatthe blood vessel cells are transferred on the tissue to be contacted ina desired form and the blood vessels are artificially formed by makingthe tissue to be contacted as an anchorage by contacting directly thebase material with the blood vessel cells adhered to the blood vesselcell adhesion portion with the tissue such as skin.

Furthermore, after it was formed in a targeted pattern on the bloodvessel cell adhesion portion, artificial blood vessels can be formeddirectly on the cell culturing layer by adding on the culture medium thegrowth factor for promoting to make the blood vessel cells into bloodvessels such as bFGF and VEGF. To stably perform the formation of theblood vessels of the blood vessel cells, however, it is desired that theformer method using an anchorage is applied.

B. Method of Manufacturing Artificial Tissue

Next, a method of manufacturing an artificial tissue of the presentinvention will be explained below. A method of manufacturing anartificial tissue of the present invention is characterized in that ituses artificial blood vessels manufactured according to the “A method ofmanufacturing an artificial blood vessel”. According to the presentinvention, since the artificial blood vessels manufactured by theabove-described method of manufacturing are used, it can be made into anartificial tissue having the blood vessels formed in a similar patternform with the blood vessel pattern existing in the living tissues.Therefore, it becomes possible that the nutrients are supplied fromthese artificial vessels to the respective tissues in the artificialtissues manufactured by the present invention, and it can be made anartificial tissue that can be used in a variety of uses.

As a method of manufacturing an artificial tissue in the presentinvention, for example, if it has the above-described artificial bloodvessels, it is not particularly limited. For example, it can be formedby the blood vessels existing in the living body and the other cells orthe like to be an organ such as kidney, or liver and a variety of kindsof organs.

As a method of manufacturing an artificial tissue in the presentinvention, for example, a method in which cells other than the bloodvessels forming the artificial tissue are cultured on the culture mediumand made into a tissue and the blood vessels are disposed on thesecells, a method in which the above-described artificial blood vesselsare disposed on the culture medium, the above-described cells arecultured on the culture medium and made into a tissue are listed. In thepresent invention, it may be made into an artificial tissue by combininga plurality of the layers formed in this way.

Moreover, as a cell used at this time, if it is a cell that becomesactive receiving the supply such as oxygen and nutrients from theabove-described blood vessels and is capable of constituting theartificial tissue, it is not particularly limited. For example, cellspecies having the metabolic functions such as hepatic parenchymal cell,and Langerhans' cell, cell species of information transmission systemsuch as brain cells and nerve cells are listed. As the above-describedcell used for manufacturing an artificial tissue of the presentinvention, it is not limited to one species, and may be cells bycombining a plurality of kinds of cells.

The culture medium for culturing the above-described cell or the like isappropriately selected by the targeted cell. Since the culture mediumgenerally used for the culturing cells can be used, the detailedexplanation is not repeated here.

C. Photomask

Next, a photomask of the present invention will be explained below. Aphotomask of the present invention is characterized in that it has ablood vessel pattern comprising a two-dimensional pattern constituted bythe line width in which a vascular endothelial cell is a tubular form.

According to the present invention, since a photomask has a patterncomprising the line width in which a vascular endothelial cell is in atubular form, the blood vessels formed in the above-described patterncan be formed using this photomask, for example, by patterning the bloodvessel cell adhesion layer into the pattern form and culturing avascular endothelial cell on the cell adhesion layer and making it in atubular form. Therefore, the blood vessels having a variety of patternscan be formed using this photomask, and the blood vessels having apattern similar to the form of the blood vessels of the living tissuecan be formed by utilizing it, for example, for exposure of the cellculturing layer as explained in the process for forming a blood vesselcell culturing pattern of the above-described “A method of manufacturingan artificial blood vessel”.

Here, as for the line width in which the vascular endothelial cell is ina tubular form, although it depends upon the kind of a vascularendothelial cell, it is preferably to be usually in the range from 20 μmto 100 μm, and more preferably to be in the range from 40 μm to 80 μm,and particularly preferably to be in the range from 50 μm to 70 μm. Bymaking it in the above-described ranges, it is because a vascularendothelial cell can be efficiently made into a tubular form. In thepresent invention, at the time when it is exposed by utilizing thephotomask, the pattern of the pattern might be enlarged or reduced andto be exposed. In this case, it is preferable that the line width of theabove-described pattern formed on the photomask is not limited in theabove-described ranges, however, it is preferable that the line width ofthe photomask is determined so that the line width of the blood vesselpattern of the projected image is in the above-described range.

Moreover, in a photomask of the present invention, for example, it maybe available that the pattern of the blood vessels is formed to be thelight shielding portion, and it may be also available that it is formedsuch that the pattern of the blood vessels becomes the opening portion.

Still further, as for the blood vessel pattern comprising atwo-dimensional pattern constituted with the above-described line width,if it is a blood vessel pattern represented in two-dimension, it is notparticularly limited. For example, it can be made a blood vessel patternobserved on a predetermined cross-section of the living tissue. In thecase where the pattern of the blood vessels is made in a blood vesselpattern of the living tissue, it may be formed such that the bloodvessel pattern of the living tissue and the pattern formed on thephotomask are the same to each other. Alternatively, for example, inorder to make the formation of the blood vessels easy, the pattern ofthe respective blood vessels formed on the photomask may be adjusted.Still further, it may be the pattern in which the necessary form of theblood vessels has been added, deleted and so on. As for theabove-described adjustment, for example, it can be made the adjustmentas explained in the process for adjusting the blood vessel formationpattern of “A method of manufacturing an artificial blood vessel”.

The above-described living tissues denote to tissues existing in theliving body and these are tissues formed with the blood vessel cells andthe other cells and these are organs. For example, they include an organsuch as kidney or liver and the respective kinds of organs includingblood vessels such as ocular fundus or skin.

In the present invention, if it is possible that the photomask asdescribed above is formed, the method is not particularly limited. Sincea forming method of a photomask, and its materials or the like aresimilar to the method or the material used in a general photomask, thedetailed explanation is not repeated here.

D. Artificial Blood Vessel

Next, artificial blood vessel of the present invention will be explainedbelow. Artificial blood vessels of the present invention ischaracterized in that it has a blood vessel pattern formed by thetwo-dimensional pattern constituted with the line width in which avascular endothelial cell is in a tubular form.

According to the present invention, since the artificial blood vessel isformed by the above-described pattern, for example, it can be made anartificial blood vessel having a pattern similar to the blood vessels inthe living tissue and it can be made into the blood vessels for playingthe functions similar to the blood vessels existing in the living tissueby for example disposing it at the infarct site of the micro bloodvessels or in an artificial tissue.

Here, as for the line width in which a vascular endothelial cell is in atubular form, although it depends upon the kind of a vascularendothelial cell constituting the artificial blood vessel of the presentinvention, it is usually in the range from 20 μm to 100 μm, preferablyin the range from 40 μm to 80 μm, and particularly preferably in therange from 50 μm to 70 μm. It is because owing to this, a vascularendothelial cell is in a tubular form, and the formation of artificialblood vessel of the present invention becomes easy.

Moreover, as for a blood vessel pattern formed by a two-dimensionalpattern constituted by the above-described line width, it can be madeinto a blood vessel pattern in a tubular form by culturing a vascularendothelial cell culture in a pattern form of the above-describedpattern form. For example, it can be made into a blood vessel patternobserved on a predetermined cross-section of the living tissue. At thistime, the above-described blood vessel pattern can be made into thepattern form same to the blood vessels existing in the living tissue,however, for example, it can be also made into a pattern in which theform of the blood vessel such as the diameter or the position of theblood vessel existing in the living tissue has been adjusted; or apattern in which the blood vessel for introducing the blood from theblood vessel existing in the artificial tissue or the living body, orthe blood vessels for exhausting has been formed. As for theabove-described adjustment, for example, it can be made the adjustmentas explained in the process for adjusting a blood vessel formationpattern of “A method of manufacturing an artificial blood vessel”.

The above-described living tissues denote to tissues existing in theliving body and these are tissues formed by the blood vessel cells, theother cells or the like. For example, these include an organ such askidney or liver, and the respective kinds of organs including bloodvessels such ocular fundus, or skin. In the present invention, as aforming method of the above-described artificial blood vessels, it couldbe formed by the method explained in the above-described “A method ofmanufacturing an artificial blood vessel”, and as for the material usedin the formation of artificial blood vessels of the present invention,it can be made material similar to the above-described material.Accordingly, the detailed explanation is not repeated here.

E. Artificial Tissue

Next, an artificial tissue of the present invention will be explainedbelow. An artificial tissue of the present invention is characterized inthat it has the above-described artificial blood vessels in addition tothe parenchymal cell having the desired functions derived from theorgan. In the case where the block of the cells only comprisesparenchymal cell of the organ, since the carrying of nutrients andmetabolic decomposition product is not performed, the cell existingwithin the internal site is necrotized. According to the presentinvention, since it becomes possible that the tissue having theartificial blood vessel is formed in addition to the parenchymal cell ofthe organ, it can be made function without necrotizing the tissue.Therefore, according to the present invention, it can be made into anartificial tissue that can be used for a variety of uses.

As for an artificial tissue of the present invention, if it is anartificial tissue having the above-described blood vessel, it is notparticularly limited, however, it can be made into a tissue formed bythe blood vessels existing in the living body, the other cells or thelike. For example, it can be made into organs such as kidney or liver,and a variety of kinds of organs such as ocular fundus or skin.

As for a method of manufacturing the artificial tissue, since it can bemade into a method similar to the method explained in theabove-described “B. Method of manufacturing artificial tissue”, thedetailed explanation is not repeated here.

F. Blood Vessel Cell Culturing Pattern Base Material

Next, a blood vessel cell culturing pattern base material of the presentinvention will be explained below. A blood vessel cell culturing patternbase material of the present invention comprises: a base material; acell culturing layer formed on the base material and having a patterncomprising a cell adhesion portion having adhesive properties with ablood vessel cell and a cell adhesion-inhibiting portion for inhibitingadhesion with the blood vessel cell; and a blood vessel cell adhered tothe cell adhesion portion, characterized in that the cell adhesionportion is formed in a blood vessel pattern comprising a two-dimensionalpattern constituted with a line width in which a vascular endothelialcell is in a tubular form.

As shown in FIG. 5, A blood vessel cell culturing pattern base materialof the present invention has the base material 11, the cell culturinglayer 1 having a pattern comprising the cell adhesion portion 2 and thecell adhesion-inhibiting portion 3 formed on the base material 11 in apredetermined pattern form, and the blood vessel cell 4 adhered to thecell adhesion portion 2.

According to the present invention, since the cell adhesion portion hasbeen formed on the cell culturing layer in a predetermined pattern formand the area other than that is made as the cell adhesion-inhibitingportion, blood vessel cell can be cultured in a finely processed patternonly on the cell adhesion portion. Owing to this, it can be made intoblood vessel cell culturing pattern base material capable of formingblood vessels in a targeted finely processed pattern. Still further,since the line width of the cell adhesion portion has been made into aline width such that a vascular endothelial cell is in a tubular form,it can be made into a blood vessel cell culturing pattern base materialcapable of efficiently forming the artificial blood vessel by easilymaking the cultured blood vessel cell into a tubular form.

Here, as for the line width in which a vascular endothelial cell is in atubular, it is a line width in which the blood vessel cell cultured onthe cell adhesion portion and made into the tissue becomes in a tubularform. Specifically, although it depends upon the kind of blood vesselthe cell or the like, it is usual in the range from 20 μm to 100 μm,more preferably in the 40 μm to 80 μm, and particularly preferably inthe range from 50 μm to 70 μm. It is because owing to this, artificialblood vessels are easily formed with the blood vessel cells cultured onthe cell adhesion portion.

Moreover, as for a blood vessel pattern comprising a two-dimensionalpattern constituted with the above-described line width, if it is ablood vessel pattern represented in two-dimension, it is notparticularly limited. For example, it can be made a blood vessel patternobserved on a predetermined cross-section of the living tissue. At thistime, the form of the above-described pattern form may be the same withthat of the blood vessels existing in the living tissue. Alternatively,for example, it can be also made a pattern in which a form of the bloodvessel such as the diameter or position of the blood vessels existing inthe living tissue has been adjusted, or a pattern in which the bloodvessels for introducing the blood from the blood vessels existing in theartificial tissue or the living body or the blood vessels for exhaustinghave been formed.

Here, as for a base material used for the blood vessel cell culturingpattern base material of the present invention, a cell culturing layerhaving the cell adhesion portion and the cell adhesion-inhibitingportion and blood vessel cells, they can be similar to those explainedin the item of the above-described “A method of manufacturing anartificial blood vessel”. Accordingly, the detailed explanation is notrepeated here.

If the blood vessel cell culturing pattern base material as describedabove is used, the following a vascular endothelial cell pattern basematerial characterized in can be obtained; the base material iscomprising the base material and a vascular endothelial cell provided onthe base material in such a way that can be peeled off, and the vascularendothelial cell has been formed with the line width becoming in atubular form in a pattern in which the blood vessel network has beenexpressed in two-dimension. In the above-described vascular endothelialcell pattern base material, since a vascular endothelial cell formed ina predetermined pattern form has been provided in a way that can bepeeled off, the vascular endothelial cell is peeled off from thevascular endothelial cell pattern base material, and it can be used fora variety of uses such as artificial tissue.

In the base material of a vascular endothelial cell with which theabove-described vascular endothelial cell can be peeled off, as aculture base, the vascular endothelial cell pattern base material whichdoes not damage a vascular endothelial cell and is capable of peelingoff a vascular endothelial cell is used. As the above-described culturebase, a culture base having a surface capable of holding the cell by aweak adhesive force is listed. Specifically, a culture base in which theplasma treatment for adhering the cell has been provided to apolystyrene base material, a culture base in which a material having thecell adhesion-inhibiting properties such as 2-methacryloyloxyethylphosphorylcoline or fluoroalkylsilane has been slightly introduced onthe surface of the base material are listed as examples. As theabove-described method of introducing small amount, a method ofdecomposing by a UV treatment, an ozone treatment, a plasma treatmentafter the material has been sufficiently introduced into the basematerial by an absorbing treatment or the like, or a method of coating athin layer with a solution thinly resolved in a solution and the likeare listed. As for the rate of introduction, it is different dependingupon the kind of cell to be adhered and the kind of a material which isintroduced into the base material, therefore, it is necessary to beadjusted.

Moreover, temperature-responsive polymer materials such aspoly-N-isopropylacryl amide, in which a material that is hydrophobic inthe environment at the temperature of the phase transition temperatureor higher and have cell adhesion properties, while it is hydrophilic atthe temperature of the phase transition temperature or lower and losethe cell adhesion properties, has been polymerized on the base materialof the high molecular polymer, a glass or the like, can be also used.

The present invention is not limited to the above-described embodiment.The above-described embodiments have been exemplified, and anyembodiment which has the constitution substantially the same with thetechnical idea described in the scope of the present invention andexerts a similar action effect is included in the technical scope of thepresent invention.

EXAMPLES

Hereinafter, Examples are shown and the present invention will befurther specifically explained below.

Example 1

<Photomask>

The photograph of human ocular fundus was shot and the blood vesselimage in the living body was obtained. This image was installed by ascanner into the computer. Next, the image installed was binarized byScion Image, the noise was removed by median filter treatment, and theedge enhancement was performed. The blood vessel formation pattern inthe living body adjusted in the most suitable width of the blood vesselcell adhesion for the blood vessel formation was prepared by extractingthe line pattern from the binarized blood vessel image, victorizing andfurther integrating the line width into 60 μm.

Further, the prepared blood vessel formation pattern was vectrized againand, similar to the usual photomask preparation procedure, a photomaskhaving a desired pattern was prepared by a laser drawing machine. At thetime when the photomask was prepared, the blood vessel portion was madea transparent portion, the portion other than the blood vessel was madethe light shielding portion.

Example 2

<Blood Vessel Cell Culturing Pattern Base Material>

Mixed for 12 hours were 1.5 g of fluoroalkylsilane TSL8233 (GE ToshibaSilicone Co., Ltd.), 5.0 g of tetramethoxysilane TSL8114 (GE ToshibaSilicone Co., Ltd.), 2.4 g of 5.0×10⁻³ NHCl, and the resultant wasdiluted into 10-fold with isopropyl alcohol. Next, 2.0 g of thissolution was coated on the base material of soda glass having the sizeof 10 cm×10 cm using a spin coater at 1000 rpm for 5 seconds, the basematerial was dried at 150° C. for 10 minutes and made it into a bloodvessel cell adhesion-inhibiting layer.

Next, 3.0 g of titanium oxide sol solution which has been diluted into3-fold with isopropyl alcohol (Ishihara Sangyo Kaisha, Ltd. STK-03) wasmade composition for the photocatalyst-containing layer. The compositionfor the photocatalyst-containing layer was coated at 700 rpm for 3seconds using a spin coater on the pattern surface of the quartzphotomask prepared by a method similar to Example 1, and a photomaskhaving a transparent photocatalyst-containing layer was prepared byperforming the drying treatment at 150° C. for 10 minutes.

These were disposed such that the interval between the surface of thephotocatalyst-containing layer of the photomask and the surface of theblood vessel cell adhesion-inhibiting layer of the base material becomes3 μm, ultraviolet ray exposure was performed for a predetermined timeperiod at 4 J/cm² using a mercury lamp (wavelength: 365 nm) from thephotomask side, and a cell culturing pattern having the cell adhesionportion having the width of 60 μm was obtained.

<Blood Vessel Cell Culturing Process>

As a culture cell, a vascular endothelial cell derived from bovinecarotid artery (see Onodera M, Morita I, Mano Y, Murota S: Differentialeffects of nitric oxide on the activity of prostaglandin endoperoxide hsynthase-1 and -2 in vascular endothelial cells, Prostag Leukotress 62:161-167, 2000) of 5-generation to 20-generation of the passage numberwere used.

A vascular endothelial cell derived from bovine carotid artery in theconfluent state in 10 cm dish was treated with 0.05% tripsin-EDTA, andpeeled off. The number of cells was examined using Coulter Counter ZM(trademark), and made it 10⁶ pieces/mL. The base material having thecell culturing pattern previously prepared was sterilized using anautoclave. The base material with the above-described cell culturingpattern formed has been inputted in the culture dish (Heraeus Quadriprem(trademark) 76×26 mm, 1976 mm²) containing the culture solution (5%bovine fetal serum containing MEM culture medium), the above-describedendothelial cells were disseminated at 10⁶ pieces/5 mL per 1 well, andincubated for 24 hours using CO₂ cell culture device. Owing to this, ablood vessel cell culturing pattern base material in which blood vesselcells were adhered in the cell culturing pattern form was obtained.

Example 3

To the blood vessel cell culturing pattern base material to which theblood vessel cells were adhered in a pattern form prepared in Example 2,2 mL of GFR matrigel (manufactured by Becton, Dickinson and Company) asan anchorage for making blood vessel tissue of the blood vessel cellswas made in contact with the blood vessel cell adhesion surface side ofthe base material, and heated at 37° C. After the gel was solidified,the base material and the gel were inputted in the CO₂ cell culturedevice in the presence of the culture solution, and the blood vesselcells were made into a tissue.

After 24 hours, when the blood vessel tissue prepared was observed by aretardation microscope, and after cell staining, immunostaining wereperformed, it was observed by a fluorescence microscope, the formationof a tubular tissue having an internal cavity was admitted.

Example 4

The blood vessel cell culturing pattern base material to which the bloodvessel cells have adhered in a pattern form by a similar procedure withExample 2 was immersed on the culture medium in which carbocyaninefluorescence pigment (DiI, manufactured by Invitrogen, Co., Ltd.) wasresolved at the concentration of 10 μg/mL with respect to 5% bovinefetal serum containing MEM culture medium, and cultured at 37° C. forone hour. Subsequently, the blood vessel cell culturing pattern basematerial was returned to 5% bovine fetal serum containing MEM culturemedium.

(Cell Introduction to Living Tissue)

The immunodeficient mouse was anesthetized, the back was cut, and thebase material to which the prepared blood vessel cells are aligned andadhered was subcutaneously transplanted. The transplanted portion wassutured. The transplanted portion was cut again at 3 day after it wassutured, then, FITC-Dextran resolved solution was injected from the tailvein and the blood was stained. The sacrificial death operation wasperformed to the mouse and DiI and FITC of the transplanted tissueportion were observed by a confocal laser scanning microscope.

As a result of the observation, the facts that the fluorescence-labeledblood vessel cells formed the structure of the blood vessels in thetransplanted tissue portion and the blood containing a fluorescencepigment communicated within the structure of the blood vessels wereadmitted.

Example 5

With respect to the Schale in which 0.5 mL of matrigel was dropped, thecell adhesion surface of the blood vessel cell culturing pattern basematerial to which the blood vessel cells have adhered in a patternprepared in Example 2 was superposed so that the cell adhesion surfaceis made in contact to the gel, and the gel was heated to 37° C. andsolidified. The base material and the gel were inputted in the CO₂ cellculture device in the presence of the culture solution and cultured for24 hours, and the blood vessel cells were made into a tissue. After theblood vessels were formed, the base material was peeled off from thegel.

In parallel to the above-described operation, the mouse liverparenchymal cell was collected, after it was stained with 10 μg/mL ofcarbocyanine fluorescence pigment (DiO, manufactured by Invitrogen, Co.,Ltd.), it was returned to 5% bovine fetal serum containing MEM culturemedium, and fluorescence stained mouse liver parenchymal cell wasobtained.

After the liver parenchymal cell was embedded in the gel bydisseminating the above-described fluorescence stained liver parenchymalcell on the gel having the artificial blood vessels, inputting thefluorescence stained liver parenchymal cell in the CO₂ cell culturedevice and culturing it for 24 hours, the tissue was peeled off from theSchale using tweezers and a tissue piece comprising a gel having theartificial blood vessels and liver parenchymal cell was prepared.

(Evaluation of Tissue)

The immunodeficient mouse was anesthetized, abdominal portion was cut, ⅓of the liver was removed, and made it into a liver function failuremodel. Next, the tissue piece prepared in the above-described operationwas transplanted in the liver portion of the mouse. The transplantedportion was sutured and the evaluation of the liver functions wasperformed by cholinesterase evaluation after 14 days, as a result ofthis, it was admitted that the liver functions were recovered to thesame level prior to the removal of the liver.

Furthermore, the liver function transplanted portion was cut again, andthe transplanted tissue was observed using a confocal laser scanningmicroscope. The transplanted liver parenchymal cell was observed by theobservation at 480 nm of the excitation wavelength. Moreover, it wasadmitted that capillary blood vessels have been formed as a vascularendothelial cell was previously patterned by the observation at 530 nmof the excitation wavelength.

Comparative Example 1

The immunodeficient mouse was anesthetized, the abdominal portion wascut, ⅓ of the liver was removed, and made it into a liver functionfailure model. The transplanted portion was sutured again withouttransplanting the tissue pieces, the evaluation of the liver functionswas performed by cholinesterase evaluation after 14 days. As a result ofthis, it was admitted that the liver functions remained was about 40%comparing to the prior to the removal of the liver.

Comparative Example 2

The mouse liver parenchymal cell was collected, after it was stainedwith 10 μg/mL of carbocyanine fluorescence pigment (DiO, manufactured byInvitrogen, Co., Ltd.), it was returned to 5% bovine fetal serumcontaining MEM culture medium, and fluorescence stained mouse liverparenchymal cell was obtained.

After the liver parenchymal cell was embedded in the gel bydisseminating the above-described fluorescence stained liver parenchymalcell on the matrigel not having the blood vessel cells, inputting thefluorescence stained liver parenchymal cell in the CO₂ cell culturedevice and culturing it for 24 hours, the tissue was peeled off from theSchale using tweezers and a gel tissue piece comprising with the liverparenchymal cell embedded was prepared.

(Evaluation of Tissue)

The immunodeficient mouse was anesthetized, abdominal portion was cut, ⅓of the liver was removed, and made it into a liver function failuremodel. Next, the tissue piece prepared in the above-described operationwas transplanted in the liver portion of the mouse. The transplantedportion was sutured and the evaluation of the liver functions wasperformed by cholinesterase evaluation after 14 days, as a result ofthis, it was admitted that the liver functions remained was about 40%comparing to the prior to the removal of the liver.

Furthermore, the liver function transplanted portion was cut again, andthe transplanted tissue was observed using a confocal laser scanningmicroscope. When the transplanted liver parenchymal cell was observed bythe observation at 480 nm of the excitation wavelength, it was admittedthat the transplanted liver parenchymal cell was scarcely seen andalmost all of the transplanted cells were necrotized.

1. A method of manufacturing an artificial blood vessel, wherein themethod comprises: preparing an image of a target living tissue;extracting a blood vessel formation image comprising only linesrepresenting the blood vessels from the image of the tissue wherein theblood vessel formation image has substantially no contours of the livingtissue or noise; adjusting the width of the lines representing the bloodvessels in the blood vessel formation image to create an adjusted bloodvessel formation image; preparing a photomask which corresponds to theadjusted blood vessel formation image, wherein the solid portions of thephotomask replicate the pattern of the adjusted blood vessel formationimage; preparing a patterning substrate which comprises a base materialand a cell culture layer formed on the base material, wherein the cellculture layer contains a cell adhesive material that has cell adhesiveproperties and is denatured by action of a photocatalyst when exposed toenergy irradiation which in turn enables a formation of a celladhesion-inhibiting portion; preparing a photocatalyst-containingsubstrate which comprises a base body and a photocatalyst-containinglayer formed on the base body and containing the photocatalyst;arranging the cell culture layer of the patterning substrate and thephotocatalyst-containing layer of the photocatalyst-containing substrateto face each other so that the cell culture layer and thephotocatalyst-containing layer actually contact each other or face eachother at a distance; arranging the photomask which corresponds to theadjusted blood vessel formation image on the base body side of thephotocatalyst-containing substrate; irradiating energy through thephotomask to the photocatalyst-containing substrate, whereby exposure ofthe unmasked portions of the photocatalyst-containing layer to theenergy irradiation activates the photocatalyst and causes denaturing ofthe cell adhesion material in corresponding regions of the cellculturing layer of the patterning substrate, wherein denaturing of thecell adhesion material results in formation of cell adhesion-inhibitingportions; thereby forming on the cell culturing layer of the patterningsubstrate a cell adhesive region in a pattern corresponding to theadjusted blood vessel formation image, and a cell adhesion-inhibitingregion in all other areas; and energy; and adhering a blood vessel cellto a cell adhesive portion in the cell culturing layer; and culturingthe blood vessel cell on the cell culturing layer to form an artificialblood vessel.
 2. A method of manufacturing an artificial tissue, whereinthe method comprises: manufacturing an artificial blood vessel accordingto claim 1; and forming an artificial tissue using the artificial bloodvessel.